09_dge_architecture

Rationale: This script performs differential gene expression (dge) and calculates the upregulated and downregulated genes in different parts of lichen thallus using Sleuth

1. Prepare data

• list samples to be included

library(sleuth)
library(tidyverse)
samples<-read.delim("../data/metadata/found_samples_in_sequences_db_thallus.csv",sep=",")

kal_dirs2 <- data.frame("sample"=samples$run_id, "part"= samples$sample_focus,
                       substrate=samples$growth_site, thallus = samples$thallus_id,
                       "path"=paste0("../analysis_and_temp_files/06_meta_mapping/kallisto_meta_mapping/",samples$run_id,"_kallisto"))  %>% filter(sample!="MP_I" & sample!="MP_II")

• define list of genes to be included: only mycobiont and only those that have read count across whole panel of >=5

full<-read.delim("../analysis_and_temp_files/08_dge_culture_lichen/counts_table.tsv",row.names=1)
d<-full %>% select(any_of(kal_dirs2$sample)) #include only lichen samples
colnames(d)

##  [1] "XBC2"   "XBA1"   "XSA2_2" "XSC1"   "XSE2"   "XBE1"   "XTA2"   "XSC2"  
##  [9] "XSA2"   "XBA2"   "XBC1"   "XTA1"   "XSA1"   "XTC2"   "XTE2"   "XBE2"  
## [17] "XMC2"

d$max<-apply(d,1,max)
min(d$max)

## [1] 0

d1<-d[which(d$max>=5),] #to remove lowly expressed genes, keep rows in which max read count across whole panel is atleast 5

# drop the max column, as its not needed anymore
raw_counts<-d1 %>% select(-max)
length(rownames(d1)) # total number of genes

## [1] 11083

length(rownames(raw_counts)) 

## [1] 11083

#make the list of selected genes
target_id<-rownames(d1)

2. Edge / Center comparison

• read in the data, including only selected genes
• modeled the DGE following the official tutorial
• Produced 7 genes upregulated in center, if we apply the thresholds of qvalue<0.05 and abs(b)>1. No genes upregulated in the edge

ec_dirs<-kal_dirs2 %>% filter(part %in% c("thallus_centre", "thallus_edge") )
ec_dirs$part <- as.factor(ec_dirs$part)

ec_so <- sleuth_prep(ec_dirs,extra_bootstrap_summary = TRUE,filter_target_id = target_id,transformation_function = function(x) log2(x + 0.1))

## Warning in check_num_cores(num_cores): It appears that you are running Sleuth from within Rstudio.
## Because of concerns with forking processes from a GUI, 'num_cores' is being set to 1.
## If you wish to take advantage of multiple cores, please consider running sleuth from the command line.

## reading in kallisto results

## dropping unused factor levels

## ..........
## normalizing est_counts
## A list of target IDs for filtering was found. Using this for filtering
## 11083 targets passed the filter
## normalizing tpm
## merging in metadata
## summarizing bootstraps
## ..........

#model that includes only confounding variables
ec_so <- sleuth_fit(ec_so, ~thallus, 'reduced') 

## fitting measurement error models
## shrinkage estimation
## 7 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_000967-T1, XANPAGTX0501_000969-T1, XANPAGTX0501_000980-T1, XANPAGTX0501_002903-T1, XANPAGTX0501_004641-T1, XANPAGTX0501_005100-T1, XANPAGTX0501_006796-T1
## computing variance of betas

#model that includes only both the confounding variable and the variable of interest
ec_so  <- sleuth_fit(ec_so, ~thallus + part, 'full')

## fitting measurement error models
## shrinkage estimation
## 7 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_000967-T1, XANPAGTX0501_000969-T1, XANPAGTX0501_000980-T1, XANPAGTX0501_002903-T1, XANPAGTX0501_004641-T1, XANPAGTX0501_005100-T1, XANPAGTX0501_006796-T1
## computing variance of betas

#compare the models
ec_so <- sleuth_lrt(ec_so, 'reduced', 'full')
ec_so <- sleuth_wt(ec_so, 'partthallus_edge')

plot_pca(ec_so, color_by = 'part', text_labels = TRUE,use_filtered=T)

ec_table <- sleuth_results(ec_so, 'partthallus_edge')
ec_sig <- ec_table %>% tibble::as_tibble() %>% 
  filter( qval <= 0.05, grepl("GTX0501",target_id) ) %>% 
  arrange(desc(b)) %>% mutate(change = if_else(b > 1, "edge", ifelse(b< -1, "centre", "low_logFC")))

table(ec_sig$change)

## 
##    centre low_logFC 
##         9       170

• Bootstrap of the two genes most overexpressed in the center. The difference isn’t massive

ec_dge<-ec_sig$target_id[ec_sig$change!="low_logFC"]
plot_bootstrap(ec_so, 
               target_id = ec_dge[7], 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ec_so, 
               target_id = ec_dge[6], 
               units = "est_counts", 
               color_by = "part")

• About genes upregulated in center, we mostly know nothing. What we do know:
  • One secreted
  • Two from lichen-enriched orthogroups
  • One of them has two InterPro domains: F-box and LRR. This makes it likely to be a part of ubiquitin protein degradation system. F-box connects to the ubiquitin ligase complex, and LRR binds to a specific protein to be degraded (seehttps://www.sciencedirect.com/science/article/pii/S0079610799000103)

library(kableExtra)
funannot2<-read.delim2("../../02_long_read_assemblies/analysis_and_temp_files/09_ortho/lichen_enriched_ortho_in_xanpa.tsv",sep="\t")

center_ec_genes<-ec_sig %>% filter(change=="centre") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
center_ec_genes %>% 
  kable(format = "html", col.names = colnames(center_ec_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_005615-T1	-1.009109	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005614-T1	-1.075863	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008007-T1	-1.155943	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002184-T1	-1.173653	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001949-T1	-1.187615	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002506-T1	-1.194636	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008856-T1	-1.278297	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_009886-T1	-1.292990	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_009927-T1	-1.619707	NA	NA	NA		FALSE	TRUE

• This is the gene the closest to being overexpressed in the edge (b value = 0.9).

plot_bootstrap(ec_so, 
               target_id = ec_sig$target_id[ec_sig$b>0.9], 
               units = "est_counts", 
               color_by = "part")

• Interestingly, it’s a putative effector, which is upregulated in lichen and matches papain inhibitor/cellulose-binding

edge_ec_genes<-ec_sig %>% filter(b>0.9) %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
edge_ec_genes %>% 
  kable(format = "html", col.names = colnames(edge_ec_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_001015-T1	0.9022441	IPR001153 Barwin domain, IPR009009 RlpA-like protein, double-psi beta-barrel domain, IPR036908 RlpA-like domain superfamily	NA	NA		TRUE	FALSE

2.2. Power analysis: simple

• Do we have enough samples for this comparison?
• Looked at the distribution of (non-corrected) p-values. The graph looks good: close to uniform, with an increase close to 0. If our test was underpowered, we would see an increase of p-vlaues along 0->1, but this is not the case

ggplot(ec_table)+geom_histogram(aes(x=pval))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

## Warning: Removed 690798 rows containing non-finite values (`stat_bin()`).

• QQ-plot also looks good

library(MKpower)
qqunif(ec_table$pval, color.line = "orange")

• However, when looking at corrected p-values (a.k.a. q-values), the graph looks bad

ggplot(ec_table)+geom_histogram(aes(x=qval))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

## Warning: Removed 690798 rows containing non-finite values (`stat_bin()`).

* Perhaps, this particular comparison is underpowered

library(MKpower)
qqunif(ec_table$qval, color.line = "orange")

3. Center / apothetia comparison

• Only 1 gene upregulated in the center compared to 118 in apothecia

ca_dirs<-kal_dirs2 %>% filter(part %in% c("thallus_centre", "apothecia") )
ca_dirs$part <- as.factor(ca_dirs$part)
ca_dirs$thallus<- as.factor(ca_dirs$thallus)

rhdf5::h5closeAll()
ca_so <- sleuth_prep(ca_dirs,extra_bootstrap_summary = TRUE,filter_target_id = target_id,transformation_function = function(x) log2(x + 0.1))

## Warning in check_num_cores(num_cores): It appears that you are running Sleuth from within Rstudio.
## Because of concerns with forking processes from a GUI, 'num_cores' is being set to 1.
## If you wish to take advantage of multiple cores, please consider running sleuth from the command line.

## reading in kallisto results

## dropping unused factor levels

## .............
## normalizing est_counts
## A list of target IDs for filtering was found. Using this for filtering
## 11083 targets passed the filter
## normalizing tpm
## merging in metadata
## summarizing bootstraps
## .............

#model that includes only confounding variables
ca_so <- sleuth_fit(ca_so, ~thallus, 'reduced') 

## fitting measurement error models
## shrinkage estimation
## 2 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_004237-T1, XANPAGTX0501_010518-T1
## computing variance of betas

#model that includes only both the confounding variable and the variable of interest
ca_so  <- sleuth_fit(ca_so, ~thallus + part, 'full')

## fitting measurement error models
## shrinkage estimation
## 3 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_004237-T1, XANPAGTX0501_010518-T1, XANPAGTX0501_010086-T1
## computing variance of betas

#compare the models
ca_so <- sleuth_lrt(ca_so, 'reduced', 'full')
ca_so <- sleuth_wt(ca_so, 'partthallus_centre')

plot_pca(ca_so, color_by = 'part', text_labels = TRUE,use_filtered=T)

ca_table <- sleuth_results(ca_so, 'partthallus_centre')
ca_sig <- ca_table %>% tibble::as_tibble() %>% 
  filter( qval <= 0.05, grepl("GTX0501",target_id) ) %>% 
  arrange(desc(b)) %>% mutate(change = if_else(b > 1, "centre", ifelse(b< -1, "apothecium", "low_logFC")))

table(ca_sig$change)

## 
## apothecium     centre  low_logFC 
##        118          1       1928

Upregulated in apothecia

• Bootstrap of the two genes most overexpressed in the apothecium. Why are the boxes so wide?

plot_bootstrap(ca_so, 
               target_id = ca_sig$target_id[which.min(ca_sig$b)], 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ca_so, 
               target_id = ca_sig$target_id[which.min(ca_sig$b[-which.min(ca_sig$b)])], 
               units = "est_counts", 
               color_by = "part")

• Both MAT genes seem upregulated, although XANPAGTX0501_002103-T1 is just below the threshold -0.99

plot_bootstrap(ca_so, 
               target_id = "XANPAGTX0501_002103-T1", 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ca_so, 
               target_id = "XANPAGTX0501_002104-T1", 
               units = "est_counts", 
               color_by = "part")

• Of the 118 upregulated in apothecia, 87 have no functional annotations
• 9 are secreted

ap_ca_genes<-ca_sig %>% filter(change=="apothecium") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
ap_ca_genes %>% 
  kable(format = "html", col.names = colnames(ap_ca_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_001605-T1	-1.002217	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004628-T1	-1.002890	IPR002156 Ribonuclease H domain, IPR012337 Ribonuclease H-like superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K03469	FALSE	FALSE
XANPAGTX0501_009621-T1	-1.004737	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008874-T1	-1.005597	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007403-T1	-1.005610	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004416-T1	-1.006355	IPR000246 Peptidase T2, asparaginase 2, IPR029055 Nucleophile aminohydrolases, N-terminal, IPR037464 Threonine aspartase 1	NA	T02	K08657	FALSE	FALSE
XANPAGTX0501_009141-T1	-1.008026	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002166-T1	-1.010246	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003282-T1	-1.010415	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006245-T1	-1.011007	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_001082-T1	-1.011584	IPR004853 Sugar phosphate transporter domain	NA	NA	K26222	FALSE	FALSE
XANPAGTX0501_002170-T1	-1.014622	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002104-T1	-1.017319	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002183-T1	-1.017368	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004494-T1	-1.018387	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007464-T1	-1.021451	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005615-T1	-1.022220	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001888-T1	-1.023478	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005658-T1	-1.031831	IPR001153 Barwin domain, IPR009009 RlpA-like protein, double-psi beta-barrel domain, IPR036908 RlpA-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002163-T1	-1.035184	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008533-T1	-1.037400	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009317-T1	-1.039048	IPR008979 Galactose-binding-like domain superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_005061-T1	-1.045582	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_001947-T1	-1.046488	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004580-T1	-1.050842	IPR007292 Nuclear fusion protein Kar5	NA	NA		FALSE	FALSE
XANPAGTX0501_006974-T1	-1.052396	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002057-T1	-1.052723	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006203-T1	-1.055462	IPR001841 Zinc finger, RING-type, IPR002867 IBR domain, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR017907 Zinc finger, RING-type, conserved site	NA	NA		FALSE	FALSE
XANPAGTX0501_002184-T1	-1.055941	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001330-T1	-1.062267	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004493-T1	-1.069215	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005597-T1	-1.070417	IPR006421 Glycogen debranching enzyme, metazoa, IPR008928 Six-hairpin glycosidase superfamily, IPR012341 Six-hairpin glycosidase-like superfamily, IPR017853 Glycoside hydrolase superfamily, IPR029436 Eukaryotic glycogen debranching enzyme, N-terminal domain, IPR032788 Glycogen debranching enzyme, central domain, IPR032790 Glycogen debranching enzyme, C-terminal, IPR032792 Glycogen debranching enzyme, glucanotransferase domain	GH133	NA	K01196	FALSE	FALSE
XANPAGTX0501_008628-T1	-1.071911	IPR023346 Lysozyme-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_003775-T1	-1.074091	IPR003100 PAZ domain, IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032473 Protein argonaute, Mid domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K11593	FALSE	FALSE
XANPAGTX0501_000647-T1	-1.082215	IPR000571 Zinc finger, CCCH-type	NA	NA		FALSE	FALSE
XANPAGTX0501_006255-T1	-1.084624	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010303-T1	-1.086556	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004891-T1	-1.088838	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000681-T1	-1.089087	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001010-T1	-1.089790	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004217-T1	-1.090288	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA	K24990	FALSE	FALSE
XANPAGTX0501_001436-T1	-1.091349	IPR002048 EF-hand domain, IPR011992 EF-hand domain pair, IPR018247 EF-Hand 1, calcium-binding site	NA	NA		FALSE	FALSE
XANPAGTX0501_006910-T1	-1.092752	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007871-T1	-1.093262	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000005-T1	-1.100476	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009139-T1	-1.106326	IPR001876 Zinc finger, RanBP2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_001333-T1	-1.109653	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_003212-T1	-1.110801	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_008009-T1	-1.110995	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006016-T1	-1.111020	IPR008928 Six-hairpin glycosidase superfamily, IPR031335 Glycosyl hydrolase family 63, C-terminal	GH63	NA		FALSE	FALSE
XANPAGTX0501_008008-T1	-1.111120	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006330-T1	-1.113100	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004104-T1	-1.113445	IPR001214 SET domain, IPR011990 Tetratricopeptide-like helical domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001362-T1	-1.113981	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001951-T1	-1.118810	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005630-T1	-1.120143	IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_008534-T1	-1.122508	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000025-T1	-1.123779	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004950-T1	-1.124212	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002522-T1	-1.132906	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003572-T1	-1.135075	IPR001087 GDSL lipase/esterase, IPR036514 SGNH hydrolase superfamily	CE16	NA		TRUE	FALSE
XANPAGTX0501_007067-T1	-1.135736	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007234-T1	-1.142869	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007262-T1	-1.143084	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010203-T1	-1.143084	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005279-T1	-1.148052	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010380-T1	-1.148975	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_010007-T1	-1.149075	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009615-T1	-1.156978	IPR035940 CAP superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_002172-T1	-1.161670	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003209-T1	-1.170208	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008041-T1	-1.181328	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_001910-T1	-1.190522	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009318-T1	-1.192876	IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_004521-T1	-1.198724	IPR008271 Serine/threonine-protein kinase, active site, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001612-T1	-1.207043	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010304-T1	-1.207964	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001538-T1	-1.211841	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009602-T1	-1.212068	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008007-T1	-1.213991	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008782-T1	-1.216240	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006951-T1	-1.222177	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005402-T1	-1.225284	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001414-T1	-1.228686	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000194-T1	-1.233329	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR013780 Glycosyl hydrolase, all-beta, IPR017853 Glycoside hydrolase superfamily	GH13	NA	K01176	FALSE	FALSE
XANPAGTX0501_005766-T1	-1.254576	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009582-T1	-1.264645	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005735-T1	-1.281286	IPR018824 Conidiation-specific protein 6	NA	NA		FALSE	FALSE
XANPAGTX0501_008331-T1	-1.288651	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001611-T1	-1.298257	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007900-T1	-1.308741	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_008090-T1	-1.312749	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002182-T1	-1.314625	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009619-T1	-1.314627	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008284-T1	-1.314835	IPR029044 Nucleotide-diphospho-sugar transferases	GT2_Glyco_tranf_2	NA		FALSE	FALSE
XANPAGTX0501_004520-T1	-1.321604	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001963-T1	-1.324243	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001960-T1	-1.329881	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000193-T1	-1.344509	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR017853 Glycoside hydrolase superfamily	GH13	NA		FALSE	FALSE
XANPAGTX0501_001967-T1	-1.356557	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001063-T1	-1.359981	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_001856-T1	-1.377390	IPR037176 Osmotin/thaumatin-like superfamily	NA	NA		TRUE	TRUE
XANPAGTX0501_002506-T1	-1.384452	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006327-T1	-1.392539	IPR018466 Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	NA	NA		TRUE	FALSE
XANPAGTX0501_007397-T1	-1.396019	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007872-T1	-1.413947	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010596-T1	-1.414271	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008420-T1	-1.415724	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000892-T1	-1.427350	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000378-T1	-1.444983	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003211-T1	-1.502300	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010280-T1	-1.533833	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005814-T1	-1.541250	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009786-T1	-1.554050	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009926-T1	-1.646262	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004966-T1	-1.664164	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008109-T1	-2.657633	IPR004143 Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL), catalytic domain	NA	NA	K03800	FALSE	FALSE
XANPAGTX0501_001156-T3	-3.910405	IPR003822 Paired amphipathic helix, IPR013194 Histone deacetylase interacting domain, IPR031693 Sin3, C-terminal, IPR036600 Paired amphipathic helix superfamily	NA	NA	K11644	FALSE	FALSE

• Selected only genes that are potentially multicellularity related

mult_list<-read.delim2("../analysis_and_temp_files/08_dge_culture_lichen/multicellularity.txt")
#select genes that come from one of the categories of interest
ips_df <-funannot2 %>% select(TranscriptID,InterPro_new) %>% 
  mutate(InterPro_new = strsplit(InterPro_new, ", I")) %>%
        unnest(InterPro_new) %>% mutate(InterPro_new=str_replace(InterPro_new,"^PR","IPR")) %>%
  mutate(short_term = substr(InterPro_new, 1,40))

ips_selected<-ips_df %>% mutate(ID=gsub( " .*$", "", InterPro_new)) %>% inner_join(mult_list,relationship = "many-to-many") %>% select(-c(InterPro_new,short_term))

## Joining with `by = join_by(ID)`

ko_cazy_selected<-funannot2 %>% select(TranscriptID,KO,CAZyme_new) %>% 
  pivot_longer(-TranscriptID,names_to = "Annotation",values_to = "ID") %>% inner_join(mult_list,relationship = "many-to-many") %>% select(-Annotation)

## Joining with `by = join_by(ID)`

mult_gene_table<-rbind(ips_selected,ko_cazy_selected)  

mult_gene_table_ap<-mult_gene_table %>% inner_join(ca_sig %>% filter(change=="apothecium") %>% select(target_id,b),by=c("TranscriptID"="target_id")) %>% arrange(Function)

mult_gene_table_ap %>% 
  kable(format = "html", col.names = colnames(mult_gene_table_ap)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

TranscriptID	ID	Annotation_type	Description	Function	Function_type	b
XANPAGTX0501_005597-T1	IPR032788	IPR	Glycogen debranching enzyme, central domain	Carbohydrate storage	Fungal multicellularity	-1.070417
XANPAGTX0501_000647-T1	IPR000571	IPR	Zinc finger, CCCH-type	Cell division, proliferation and growth	Fungal multicellularity	-1.082215
XANPAGTX0501_003775-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.074091
XANPAGTX0501_004104-T1	IPR001214	IPR	SET domain	Cell division, proliferation and growth	Fungal multicellularity	-1.113445
XANPAGTX0501_004628-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.002890
XANPAGTX0501_005735-T1	IPR018824	IPR	Conidiation-specific protein 6	Cell surface and cell wall proteins	Fungal multicellularity	-1.281286
XANPAGTX0501_006327-T1	IPR018466	IPR	Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	Cell wall biosynthesis	Fungal multicellularity	-1.392539
XANPAGTX0501_001856-T1	IPR037176	IPR	Osmotin/thaumatin-like superfamily	Cell wall remodeling	Fungal multicellularity	-1.377390
XANPAGTX0501_003212-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.110801
XANPAGTX0501_006203-T1	IPR017907	IPR	Zinc finger; RING-type; conserved site	Protein ubiquitination	Fungal multicellularity	-1.055462
XANPAGTX0501_010380-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.148975
XANPAGTX0501_004217-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.090288
XANPAGTX0501_003775-T1	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.074091
XANPAGTX0501_006203-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.055462

• Other notable proteins:
  • Another protein from ubiquitin degrading system with a F-box and LRR domains
  • Multiple transcription factors with different Zinc finger domains and IPR000210 BTB/POZ domain
  • IPR031693 Sin3, C-terminal, which is a transcriptional repressor
  • IPR018824 Conidiation-specific protein 6, function unknown
  • Secreted IPR035940 CAP superfamily protein (egulation of extracellular matrix and branching morphogenesis, either proteases or protease inhibitors)
  • Several GHs
• InterPro enrichement analysis

###make table with IPS annotations
ips_df <-funannot2 %>% select(TranscriptID,InterPro_new) %>% 
  mutate(InterPro_new = strsplit(InterPro_new, ", I")) %>%
        unnest(InterPro_new) %>% mutate(InterPro_new=str_replace(InterPro_new,"^PR","IPR")) %>%
  mutate(short_term = substr(InterPro_new, 1,40))

ips_data <- list(
    term2protein = data.frame(
                        term = ips_df$InterPro_new,
                        gene = ips_df$TranscriptID
                        ),
    term2name = data.frame(
                        term = ips_df$InterPro_new,
                        name = ips_df$short_term
                        ),
    
    universe = unique(as.character(ips_df$TranscriptID))
)

###enrichment analysis
enrich<-clusterProfiler::enricher(ap_ca_genes$target_id,
    pAdjustMethod = "none",
    minGSSize = 1,
    maxGSSize = 2000,
    qvalueCutoff = 1,
    universe=ips_data$universe,
    TERM2GENE=ips_data$term2protein,
    TERM2NAME=ips_data$term2name)

enrich_pairwise<-enrichplot::pairwise_termsim(enrich)
enrichplot::emapplot(enrich_pairwise)

The only gene showed as upregulated in center.

• This gene is expressed in most samples, but there is a high level of heterogeneity across different lichen individuals, obscuring the pattern of it being more highly expressed in the center

plot_bootstrap(ca_so, 
               target_id = ca_sig$target_id[which.max(ca_sig$b)], 
               units = "est_counts", 
               color_by = "part")

• This is the second, which is just below the threshold (b = 0.98)

plot_bootstrap(ca_so, 
               target_id = "XANPAGTX0501_004633-T1", 
               units = "est_counts", 
               color_by = "part")

• Nothing is known about either!

ce_ca_genes<-ca_sig %>% filter(b>0.9) %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
ce_ca_genes %>% 
  kable(format = "html", col.names = colnames(ce_ca_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_006303-T1	1.3084877	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004633-T1	0.9765075	NA	NA	NA		FALSE	FALSE

3.2. Power analysis: simple

• Do we have enough samples for this comparison?
• Looked at the distribution of corrected p-values (a.k.a. q-values). The graph looks good: close to uniform, with an increase close to 0. If our test was underpowered, we would see an increase of p-vlaues along 0->1, but this is not the case

ggplot(ca_table)+geom_histogram(aes(x=qval))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

## Warning: Removed 690798 rows containing non-finite values (`stat_bin()`).

• QQ-plot looks fine

library(MKpower)
qqunif(ca_table$qval, color.line = "orange")

4. Edge / apothetia comparison

• Only 1 gene upregulated in the center compared to 118 in apothecia

ea_dirs<-kal_dirs2 %>% filter(part %in% c("thallus_edge", "apothecia") )
ea_dirs$part <- as.factor(ea_dirs$part)
ea_dirs$thallus<- as.factor(ea_dirs$thallus)

rhdf5::h5closeAll()
ea_so <- sleuth_prep(ea_dirs,extra_bootstrap_summary = TRUE,filter_target_id = target_id,transformation_function = function(x) log2(x + 0.1))

## Warning in check_num_cores(num_cores): It appears that you are running Sleuth from within Rstudio.
## Because of concerns with forking processes from a GUI, 'num_cores' is being set to 1.
## If you wish to take advantage of multiple cores, please consider running sleuth from the command line.

## reading in kallisto results

## dropping unused factor levels

## ...........
## normalizing est_counts
## A list of target IDs for filtering was found. Using this for filtering
## 11083 targets passed the filter
## normalizing tpm
## merging in metadata
## summarizing bootstraps
## ...........

#model that includes only confounding variables
ea_so <- sleuth_fit(ea_so, ~thallus, 'reduced') 

## fitting measurement error models
## shrinkage estimation
## computing variance of betas

#model that includes only both the confounding variable and the variable of interest
ea_so  <- sleuth_fit(ea_so, ~ thallus + part, 'full')

## fitting measurement error models
## shrinkage estimation
## 1 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_010086-T1
## computing variance of betas

#compare the models
ea_so <- sleuth_lrt(ea_so, 'reduced', 'full')
ea_so <- sleuth_wt(ea_so, 'partthallus_edge')

ea_table <- sleuth_results(ea_so, 'partthallus_edge')
ea_sig <- ea_table %>% tibble::as_tibble() %>% 
  filter( qval <= 0.05, grepl("GTX0501",target_id) ) %>% 
  arrange(desc(b)) %>% mutate(change = if_else(b > 1, "edge", ifelse(b< -1, "apothecium", "low_logFC")))

plot_pca(ea_so, color_by = 'part', text_labels = TRUE,use_filtered=T)

table(ea_sig$change)

## 
## apothecium       edge  low_logFC 
##        371         13       1772

Upregulated in apothecia

• Bootstrap of the two genes most overexpressed in the apothecium.

plot_bootstrap(ea_so, 
               target_id = ea_sig$target_id[which.min(ea_sig$b)], 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ea_so, 
               target_id = ea_sig$target_id[which.min(ea_sig$b[-which.min(ea_sig$b)])], 
               units = "est_counts", 
               color_by = "part")

• Both MAT genes seem upregulated, with b-value of -1.8

plot_bootstrap(ea_so, 
               target_id = "XANPAGTX0501_002103-T1", 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ea_so, 
               target_id = "XANPAGTX0501_002104-T1", 
               units = "est_counts", 
               color_by = "part")

• Of the 371 upregulated in apothecia, 255 have no functional annotations
• 33 are secreted

ap_ea_genes<-ea_sig %>% filter(change=="apothecium") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
ap_ea_genes %>% 
  kable(format = "html", col.names = colnames(ap_ea_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_006767-T1	-1.001121	IPR017956 AT hook, DNA-binding motif	NA	NA		FALSE	FALSE
XANPAGTX0501_001647-T1	-1.003441	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006026-T1	-1.005997	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010639-T1	-1.009995	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000350-T1	-1.010205	IPR003439 ABC transporter-like, ATP-binding domain, IPR003593 AAA+ ATPase domain, IPR013525 ABC-2 type transporter, IPR017871 ABC transporter-like, conserved site, IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA	K05681	FALSE	FALSE
XANPAGTX0501_005347-T1	-1.010680	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004759-T1	-1.011221	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006900-T1	-1.012780	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009389-T1	-1.014539	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007378-T1	-1.016970	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005621-T1	-1.020780	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010022-T1	-1.023610	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_009499-T1	-1.023663	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006945-T1	-1.030462	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000661-T1	-1.032709	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006946-T1	-1.037233	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002624-T1	-1.038299	IPR029058 Alpha/Beta hydrolase fold	NA	NA		FALSE	FALSE
XANPAGTX0501_010094-T1	-1.039693	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008594-T1	-1.040188	IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001497-T1	-1.040517	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000203-T1	-1.040557	IPR001938 Thaumatin family, IPR037176 Osmotin/thaumatin-like superfamily	GH152	NA		TRUE	FALSE
XANPAGTX0501_010087-T1	-1.040935	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003186-T1	-1.041709	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_010640-T1	-1.042747	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004003-T1	-1.043412	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000909-T1	-1.050406	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002181-T1	-1.050504	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010204-T1	-1.051517	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006142-T1	-1.056449	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_005447-T1	-1.060382	IPR024500 Domain of unknown function DUF3074	NA	NA		FALSE	FALSE
XANPAGTX0501_004484-T1	-1.063011	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000065-T1	-1.064314	IPR027921 NOP protein chaperone 1	NA	NA		FALSE	FALSE
XANPAGTX0501_006920-T1	-1.066060	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003265-T1	-1.067945	IPR007203 ORMDL family	NA	NA		FALSE	FALSE
XANPAGTX0501_002152-T1	-1.069475	IPR027417 P-loop containing nucleoside triphosphate hydrolase, IPR040632 Sulfotransferase, S. mansonii-type	NA	NA		FALSE	TRUE
XANPAGTX0501_003742-T1	-1.069824	IPR001509 NAD-dependent epimerase/dehydratase, IPR036291 NAD(P)-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_006931-T1	-1.072494	IPR013087 Zinc finger C2H2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_002657-T1	-1.073491	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005924-T1	-1.074455	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007353-T1	-1.077217	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005583-T1	-1.079779	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009580-T1	-1.091993	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004141-T1	-1.102317	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010231-T1	-1.104490	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008637-T1	-1.106264	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_000885-T1	-1.108859	IPR036305 RGS domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_003865-T1	-1.109235	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_000417-T1	-1.109737	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005025-T1	-1.115496	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000848-T1	-1.115545	IPR016137 RGS domain, IPR036305 RGS domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_006384-T1	-1.125655	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004308-T1	-1.127582	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010011-T1	-1.134493	IPR036013 Band 7/SPFH domain superfamily	NA	NA	K07192	FALSE	FALSE
XANPAGTX0501_005047-T1	-1.135514	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002639-T1	-1.136570	IPR002740 EVE domain, IPR015947 PUA-like superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_006342-T1	-1.138964	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004244-T1	-1.139706	IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005153-T1	-1.141212	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009787-T1	-1.141882	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008527-T1	-1.142627	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009812-T1	-1.143459	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006215-T1	-1.145166	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001925-T1	-1.145918	IPR001841 Zinc finger, RING-type, IPR002867 IBR domain	NA	NA		FALSE	FALSE
XANPAGTX0501_006992-T1	-1.146775	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010010-T1	-1.146789	IPR036013 Band 7/SPFH domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009594-T1	-1.149357	IPR002877 Ribosomal RNA methyltransferase, FtsJ domain, IPR029063 S-adenosyl-L-methionine-dependent methyltransferase superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005832-T1	-1.150654	IPR009836 Glycine-rich domain-containing protein-like	NA	NA		FALSE	FALSE
XANPAGTX0501_006250-T1	-1.153982	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002478-T1	-1.154555	IPR013126 Heat shock protein 70 family, IPR043129 ATPase, nucleotide binding domain	NA	NA		FALSE	FALSE
XANPAGTX0501_003033-T1	-1.155116	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002477-T1	-1.157099	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010530-T1	-1.158664	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001222-T1	-1.167774	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009738-T1	-1.170585	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_006766-T1	-1.171555	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010007-T1	-1.175289	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009438-T1	-1.175488	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003238-T1	-1.177022	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009437-T1	-1.179555	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010368-T1	-1.180001	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001509-T1	-1.181146	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001462-T1	-1.182311	IPR008775 Phytanoyl-CoA dioxygenase-like	NA	NA		FALSE	TRUE
XANPAGTX0501_005177-T1	-1.185673	IPR001313 Pumilio RNA-binding repeat, IPR011989 Armadillo-like helical, IPR016024 Armadillo-type fold, IPR033133 Pumilio homology domain, IPR033712 Pumilio, RNA binding domain	NA	NA		FALSE	FALSE
XANPAGTX0501_001839-T1	-1.189988	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009785-T1	-1.191020	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007467-T1	-1.203546	IPR013830 SGNH hydrolase-type esterase domain, IPR036514 SGNH hydrolase superfamily	CE3	NA		TRUE	FALSE
XANPAGTX0501_010638-T1	-1.203845	IPR003593 AAA+ ATPase domain, IPR003959 ATPase, AAA-type, core, IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	TRUE
XANPAGTX0501_008862-T1	-1.204663	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008420-T1	-1.205385	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010529-T1	-1.212807	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001648-T1	-1.214948	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005677-T1	-1.220573	IPR004313 Acireductone dioxygenase ARD family, IPR011051 RmlC-like cupin domain superfamily, IPR014710 RmlC-like jelly roll fold, IPR027496 Acireductone dioxygenase, eukaryotes	NA	NA	K08967	FALSE	FALSE
XANPAGTX0501_005317-T1	-1.226163	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_005370-T1	-1.231519	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000888-T1	-1.234664	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009407-T1	-1.241242	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009599-T1	-1.242828	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006143-T1	-1.243856	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000622-T1	-1.244489	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007851-T1	-1.245823	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001498-T1	-1.248272	IPR012340 Nucleic acid-binding, OB-fold	NA	NA		FALSE	FALSE
XANPAGTX0501_008005-T1	-1.251448	IPR001965 Zinc finger, PHD-type, IPR011011 Zinc finger, FYVE/PHD-type, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR019786 Zinc finger, PHD-type, conserved site, IPR019787 Zinc finger, PHD-finger	NA	NA		FALSE	FALSE
XANPAGTX0501_005952-T1	-1.253184	IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_000707-T1	-1.255320	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003775-T2	-1.260916	IPR003100 PAZ domain, IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032473 Protein argonaute, Mid domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K11593	FALSE	FALSE
XANPAGTX0501_005280-T1	-1.264296	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003264-T1	-1.264968	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_008708-T1	-1.265455	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001062-T1	-1.268232	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006095-T1	-1.272978	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009628-T1	-1.276921	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004596-T1	-1.281428	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004753-T1	-1.281897	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000281-T1	-1.282182	IPR011701 Major facilitator superfamily, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_003198-T1	-1.286713	IPR022085 Oxopyrrolidines biosynthesis cluster protein G	NA	NA		FALSE	TRUE
XANPAGTX0501_010276-T1	-1.289004	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010523-T1	-1.289004	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007934-T1	-1.291157	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002054-T1	-1.291837	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005550-T1	-1.294683	IPR001810 F-box domain, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005188-T1	-1.298616	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004102-T1	-1.299115	IPR000420 Yeast PIR protein repeat	NA	NA	K26550	TRUE	FALSE
XANPAGTX0501_002019-T1	-1.299594	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010021-T1	-1.299618	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_002159-T1	-1.301773	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_002946-T1	-1.307564	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_003790-T1	-1.311097	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_002123-T1	-1.312729	IPR000504 RNA recognition motif domain, IPR007855 RNA-dependent RNA polymerase, eukaryotic-type, IPR035979 RNA-binding domain superfamily	NA	NA	K11699	FALSE	FALSE
XANPAGTX0501_000751-T1	-1.312812	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001644-T1	-1.313601	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007232-T1	-1.314325	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006139-T1	-1.315900	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001436-T1	-1.320276	IPR002048 EF-hand domain, IPR011992 EF-hand domain pair, IPR018247 EF-Hand 1, calcium-binding site	NA	NA		FALSE	FALSE
XANPAGTX0501_006420-T1	-1.320799	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005281-T1	-1.324225	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004466-T1	-1.328900	IPR000490 Glycoside hydrolase family 17, IPR017853 Glycoside hydrolase superfamily, IPR018620 Ubiquitin 3 binding protein But2, C-terminal	NA	NA		TRUE	FALSE
XANPAGTX0501_002098-T1	-1.334348	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_007833-T1	-1.343993	IPR002889 Carbohydrate-binding WSC, IPR018535 Domain of unknown function DUF1996	NA	NA		FALSE	FALSE
XANPAGTX0501_004270-T1	-1.346903	IPR009465 Spondin, N-terminal	NA	NA		FALSE	FALSE
XANPAGTX0501_003124-T1	-1.348872	IPR001461 Aspartic peptidase A1 family, IPR001969 Aspartic peptidase, active site, IPR021109 Aspartic peptidase domain superfamily, IPR033121 Peptidase family A1 domain, IPR034164 Pepsin-like domain	NA	A01A		TRUE	FALSE
XANPAGTX0501_000938-T1	-1.358336	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007254-T1	-1.362647	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010211-T1	-1.362647	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008200-T1	-1.362879	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003864-T1	-1.363940	IPR009075 Acyl-CoA dehydrogenase/oxidase C-terminal, IPR009100 Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain superfamily, IPR013786 Acyl-CoA dehydrogenase/oxidase, N-terminal, IPR036250 Acyl-CoA dehydrogenase-like, C-terminal, IPR037069 Acyl-CoA dehydrogenase/oxidase, N-terminal domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005775-T1	-1.364250	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007473-T1	-1.375934	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007936-T1	-1.379176	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005640-T1	-1.380432	IPR013087 Zinc finger C2H2-type	NA	NA		FALSE	TRUE
XANPAGTX0501_000026-T1	-1.381814	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010180-T1	-1.383532	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010275-T1	-1.384204	IPR003593 AAA+ ATPase domain, IPR027417 P-loop containing nucleoside triphosphate hydrolase, IPR041679 DNA2/NAM7 helicase-like, C-terminal	NA	NA		FALSE	TRUE
XANPAGTX0501_006203-T1	-1.386200	IPR001841 Zinc finger, RING-type, IPR002867 IBR domain, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR017907 Zinc finger, RING-type, conserved site	NA	NA		FALSE	FALSE
XANPAGTX0501_001955-T1	-1.387192	IPR001138 Zn(2)Cys(6) fungal-type DNA-binding domain, IPR001451 Hexapeptide repeat, IPR002110 Ankyrin repeat, IPR011004 Trimeric LpxA-like superfamily, IPR018357 Hexapeptide transferase, conserved site, IPR020683 Domain of unknown function DUF3447, IPR024688 Maltose/galactoside acetyltransferase, IPR036770 Ankyrin repeat-containing domain superfamily, IPR036864 Zn(2)-C6 fungal-type DNA-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009761-T1	-1.387848	IPR001283 Cysteine-rich secretory protein-related, IPR014044 CAP domain, IPR018244 Allergen V5/Tpx-1-related, conserved site, IPR034120 None, IPR035940 CAP superfamily	NA	NA	K20412	TRUE	FALSE
XANPAGTX0501_002410-T1	-1.391729	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006114-T1	-1.393540	IPR001680 WD40 repeat, IPR015943 WD40/YVTN repeat-like-containing domain superfamily, IPR036322 WD40-repeat-containing domain superfamily	NA	NA	K12782	FALSE	FALSE
XANPAGTX0501_007365-T1	-1.397815	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009326-T1	-1.398307	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005584-T1	-1.398387	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001825-T1	-1.402807	IPR003663 Sugar/inositol transporter, IPR005828 Major facilitator, sugar transporter-like, IPR005829 Sugar transporter, conserved site, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_010380-T1	-1.404538	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_000033-T1	-1.410231	IPR002656 Acyltransferase 3 domain	NA	NA		FALSE	FALSE
XANPAGTX0501_009293-T1	-1.419596	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_004008-T1	-1.420375	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000036-T1	-1.420457	IPR006640 SprT-like	NA	NA		FALSE	FALSE
XANPAGTX0501_009581-T1	-1.427218	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008888-T1	-1.427796	IPR007527 Zinc finger, SWIM-type	NA	NA		FALSE	TRUE
XANPAGTX0501_003765-T1	-1.428029	IPR000420 Yeast PIR protein repeat	NA	NA		TRUE	FALSE
XANPAGTX0501_000697-T1	-1.443805	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_006214-T1	-1.444244	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_009795-T2	-1.444535	IPR023631 Amidase signature domain, IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_010304-T1	-1.458420	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006422-T1	-1.460727	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004978-T1	-1.460984	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_009621-T1	-1.461288	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006909-T1	-1.464054	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008863-T1	-1.466559	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002007-T1	-1.467093	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_002820-T1	-1.467192	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006246-T1	-1.468497	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007869-T1	-1.472330	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005619-T1	-1.474796	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010379-T1	-1.475645	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008874-T1	-1.479109	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006459-T1	-1.480266	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009605-T1	-1.480669	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002963-T1	-1.487093	IPR000172 Glucose-methanol-choline oxidoreductase, N-terminal, IPR007867 Glucose-methanol-choline oxidoreductase, C-terminal, IPR012132 Glucose-methanol-choline oxidoreductase, IPR036188 FAD/NAD(P)-binding domain superfamily	AA3	NA	K00108	TRUE	FALSE
XANPAGTX0501_009942-T1	-1.494661	IPR001810 F-box domain, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001951-T1	-1.498238	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004853-T1	-1.500419	IPR000719 Protein kinase domain, IPR000961 AGC-kinase, C-terminal, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_003361-T1	-1.505428	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_001820-T2	-1.510192	IPR021109 Aspartic peptidase domain superfamily, IPR033121 Peptidase family A1 domain	NA	NA		FALSE	FALSE
XANPAGTX0501_000691-T1	-1.511934	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001710-T1	-1.512838	IPR007577 Glycosyltransferase, DXD sugar-binding motif, IPR029044 Nucleotide-diphospho-sugar transferases	GT32	NA		FALSE	FALSE
XANPAGTX0501_003448-T1	-1.515924	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_000420-T1	-1.518444	IPR013087 Zinc finger C2H2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_005278-T1	-1.521181	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010051-T1	-1.529828	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000910-T1	-1.530331	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009511-T1	-1.533303	IPR000757 Glycoside hydrolase family 16, IPR001002 Chitin-binding, type 1, IPR008264 Beta-glucanase, IPR013320 Concanavalin A-like lectin/glucanase domain superfamily, IPR036861 Endochitinase-like superfamily	GH16	NA	K01216	TRUE	FALSE
XANPAGTX0501_008533-T1	-1.540302	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009737-T1	-1.540774	IPR005829 Sugar transporter, conserved site, IPR011701 Major facilitator superfamily, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009141-T1	-1.543886	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007899-T1	-1.544599	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003247-T1	-1.545289	IPR001878 Zinc finger, CCHC-type, IPR025836 Zinc knuckle CX2CX4HX4C, IPR036875 Zinc finger, CCHC-type superfamily	NA	NA	K09250	FALSE	FALSE
XANPAGTX0501_009666-T1	-1.554891	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001082-T1	-1.559541	IPR004853 Sugar phosphate transporter domain	NA	NA	K26222	FALSE	FALSE
XANPAGTX0501_005402-T1	-1.560177	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000872-T1	-1.562878	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005296-T1	-1.568716	IPR018750 Protein of unknown function DUF2306, membrane	NA	NA		FALSE	FALSE
XANPAGTX0501_004018-T2	-1.571979	IPR011009 Protein kinase-like domain superfamily, IPR016477 Fructosamine/Ketosamine-3-kinase	NA	NA		FALSE	TRUE
XANPAGTX0501_007750-T1	-1.577297	IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	FALSE
XANPAGTX0501_004416-T1	-1.578773	IPR000246 Peptidase T2, asparaginase 2, IPR029055 Nucleophile aminohydrolases, N-terminal, IPR037464 Threonine aspartase 1	NA	T02	K08657	FALSE	FALSE
XANPAGTX0501_007403-T1	-1.582309	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010598-T1	-1.582325	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009600-T1	-1.583776	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007231-T1	-1.583790	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010381-T1	-1.592206	IPR002293 Amino acid/polyamine transporter I	NA	NA	K03294	FALSE	FALSE
XANPAGTX0501_008534-T1	-1.593016	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001512-T1	-1.603338	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001061-T1	-1.603339	IPR002328 Alcohol dehydrogenase, zinc-type, conserved site, IPR011032 GroES-like superfamily, IPR013149 Alcohol dehydrogenase-like, C-terminal, IPR013154 Alcohol dehydrogenase-like, N-terminal, IPR020843 Polyketide synthase, enoylreductase domain, IPR036291 NAD(P)-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001953-T1	-1.607205	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005049-T1	-1.612235	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002057-T1	-1.616294	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008008-T1	-1.617050	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006330-T1	-1.618394	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001064-T1	-1.621988	IPR021765 Mycotoxin biosynthesis protein UstYa-like	NA	NA		FALSE	FALSE
XANPAGTX0501_009887-T1	-1.625740	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_001965-T1	-1.632952	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008628-T1	-1.639869	IPR023346 Lysozyme-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_000025-T1	-1.641669	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001362-T1	-1.647839	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003283-T1	-1.648782	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001948-T1	-1.662753	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001888-T1	-1.678493	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001950-T1	-1.678963	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007464-T1	-1.685849	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002213-T1	-1.688746	IPR000629 ATP-dependent RNA helicase DEAD-box, conserved site, IPR001650 Helicase, C-terminal, IPR011545 DEAD/DEAH box helicase domain, IPR014001 Helicase superfamily 1/2, ATP-binding domain, IPR014014 RNA helicase, DEAD-box type, Q motif, IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	FALSE
XANPAGTX0501_009316-T1	-1.697722	IPR001810 F-box domain, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009886-T1	-1.705744	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_003282-T1	-1.706541	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008426-T1	-1.707030	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001330-T1	-1.707882	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000681-T1	-1.709567	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001964-T1	-1.727590	IPR001611 Leucine-rich repeat, IPR003591 Leucine-rich repeat, typical subtype, IPR019487 RAM signalling pathway, SOG2, IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001949-T1	-1.732732	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006245-T1	-1.743213	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_003212-T1	-1.757104	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_009317-T1	-1.758003	IPR008979 Galactose-binding-like domain superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_009582-T1	-1.765103	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004217-T1	-1.766312	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA	K24990	FALSE	FALSE
XANPAGTX0501_005394-T1	-1.767362	IPR002575 Aminoglycoside phosphotransferase, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001947-T1	-1.767840	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003775-T1	-1.770114	IPR003100 PAZ domain, IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032473 Protein argonaute, Mid domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K11593	FALSE	FALSE
XANPAGTX0501_008419-T1	-1.778819	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004950-T1	-1.782824	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002170-T1	-1.786506	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000893-T1	-1.791529	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004628-T1	-1.792127	IPR002156 Ribonuclease H domain, IPR012337 Ribonuclease H-like superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K03469	FALSE	FALSE
XANPAGTX0501_007343-T1	-1.792140	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004494-T1	-1.792536	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010117-T1	-1.793767	IPR002110 Ankyrin repeat, IPR011009 Protein kinase-like domain superfamily, IPR016477 Fructosamine/Ketosamine-3-kinase, IPR020683 Domain of unknown function DUF3447, IPR036770 Ankyrin repeat-containing domain superfamily	NA	NA	K15523	FALSE	FALSE
XANPAGTX0501_006449-T1	-1.796457	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000005-T1	-1.801160	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005658-T1	-1.801278	IPR001153 Barwin domain, IPR009009 RlpA-like protein, double-psi beta-barrel domain, IPR036908 RlpA-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_003213-T1	-1.804291	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_001954-T1	-1.808672	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004580-T1	-1.809609	IPR007292 Nuclear fusion protein Kar5	NA	NA		FALSE	FALSE
XANPAGTX0501_002103-T1	-1.810531	IPR036910 High mobility group box domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005597-T1	-1.816345	IPR006421 Glycogen debranching enzyme, metazoa, IPR008928 Six-hairpin glycosidase superfamily, IPR012341 Six-hairpin glycosidase-like superfamily, IPR017853 Glycoside hydrolase superfamily, IPR029436 Eukaryotic glycogen debranching enzyme, N-terminal domain, IPR032788 Glycogen debranching enzyme, central domain, IPR032790 Glycogen debranching enzyme, C-terminal, IPR032792 Glycogen debranching enzyme, glucanotransferase domain	GH133	NA	K01196	FALSE	FALSE
XANPAGTX0501_005613-T1	-1.817996	IPR003615 HNH nuclease	NA	NA		FALSE	TRUE
XANPAGTX0501_006016-T1	-1.831989	IPR008928 Six-hairpin glycosidase superfamily, IPR031335 Glycosyl hydrolase family 63, C-terminal	GH63	NA		FALSE	FALSE
XANPAGTX0501_002104-T1	-1.835003	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007067-T1	-1.839308	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001612-T1	-1.846971	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000398-T1	-1.847702	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004493-T1	-1.857771	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007244-T1	-1.858539	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000610-T1	-1.868687	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001333-T1	-1.870374	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_009927-T1	-1.870484	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007870-T1	-1.874070	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004104-T1	-1.878122	IPR001214 SET domain, IPR011990 Tetratricopeptide-like helical domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005631-T1	-1.885566	IPR023631 Amidase signature domain, IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009786-T1	-1.894942	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002185-T1	-1.897085	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002163-T1	-1.904022	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006255-T1	-1.904541	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001643-T1	-1.910689	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_010222-T1	-1.912114	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002166-T1	-1.917720	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009926-T1	-1.919959	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000194-T1	-1.934433	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR013780 Glycosyl hydrolase, all-beta, IPR017853 Glycoside hydrolase superfamily	GH13	NA	K01176	FALSE	FALSE
XANPAGTX0501_000647-T1	-1.940161	IPR000571 Zinc finger, CCCH-type	NA	NA		FALSE	FALSE
XANPAGTX0501_001956-T1	-1.941094	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008090-T1	-1.954350	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002165-T1	-1.959472	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005620-T1	-1.973360	IPR001841 Zinc finger, RING-type, IPR004331 SPX domain, IPR011016 Zinc finger, RING-CH-type, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR017907 Zinc finger, RING-type, conserved site	NA	NA		FALSE	TRUE
XANPAGTX0501_004520-T1	-1.985803	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001605-T1	-1.990522	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001538-T1	-1.994056	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001414-T1	-1.994528	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005048-T1	-1.997677	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000604-T1	-1.997943	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001958-T1	-1.998637	IPR001356 Homeobox domain, IPR009057 Homeobox-like domain superfamily, IPR017970 Homeobox, conserved site	NA	NA		FALSE	FALSE
XANPAGTX0501_006974-T1	-2.000740	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009615-T1	-2.000853	IPR035940 CAP superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_001952-T1	-2.002699	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008041-T1	-2.016189	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_005630-T1	-2.027229	IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002522-T1	-2.030035	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006910-T1	-2.032529	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001967-T1	-2.032808	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003572-T1	-2.044217	IPR001087 GDSL lipase/esterase, IPR036514 SGNH hydrolase superfamily	CE16	NA		TRUE	FALSE
XANPAGTX0501_010303-T1	-2.054768	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005279-T1	-2.055973	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009139-T1	-2.056685	IPR001876 Zinc finger, RanBP2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_004891-T1	-2.057500	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001910-T1	-2.062241	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007873-T1	-2.067522	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007871-T1	-2.081123	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005615-T1	-2.092197	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009318-T1	-2.097684	IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005735-T1	-2.102015	IPR018824 Conidiation-specific protein 6	NA	NA		FALSE	FALSE
XANPAGTX0501_007872-T1	-2.105800	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001611-T1	-2.125697	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005814-T1	-2.129685	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001859-T1	-2.142380	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007262-T1	-2.149005	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010203-T1	-2.149005	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_008856-T1	-2.160218	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_005061-T1	-2.166875	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_002183-T1	-2.174606	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002182-T1	-2.180402	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002763-T1	-2.184556	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009619-T1	-2.203533	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005766-T1	-2.223453	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004414-T1	-2.230880	IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	TRUE
XANPAGTX0501_006957-T1	-2.252415	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005614-T1	-2.252890	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001960-T1	-2.264721	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010596-T1	-2.271872	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000193-T1	-2.283992	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR017853 Glycoside hydrolase superfamily	GH13	NA		FALSE	FALSE
XANPAGTX0501_004521-T1	-2.284391	IPR008271 Serine/threonine-protein kinase, active site, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002172-T1	-2.285092	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008331-T1	-2.302060	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002184-T1	-2.304675	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004966-T1	-2.317059	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001959-T1	-2.362237	IPR000095 CRIB domain, IPR000719 Protein kinase domain, IPR008271 Serine/threonine-protein kinase, active site, IPR011009 Protein kinase-like domain superfamily, IPR017441 Protein kinase, ATP binding site, IPR033923 p21 activated kinase binding domain, IPR036936 CRIB domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001063-T1	-2.369606	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_008284-T1	-2.429045	IPR029044 Nucleotide-diphospho-sugar transferases	GT2_Glyco_tranf_2	NA		FALSE	FALSE
XANPAGTX0501_008782-T1	-2.431407	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001966-T1	-2.436706	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003209-T1	-2.437600	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008009-T1	-2.444813	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001962-T1	-2.456164	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008007-T1	-2.479715	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007397-T1	-2.486783	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000378-T1	-2.490902	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009602-T1	-2.500879	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006951-T1	-2.507920	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002164-T1	-2.527734	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001856-T1	-2.592477	IPR037176 Osmotin/thaumatin-like superfamily	NA	NA		TRUE	TRUE
XANPAGTX0501_007900-T1	-2.636563	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006327-T1	-2.649849	IPR018466 Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	NA	NA		TRUE	FALSE
XANPAGTX0501_002506-T1	-2.726475	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010050-T2	-2.757580	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003211-T1	-2.931999	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007239-T1	-3.420906	IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	FALSE
XANPAGTX0501_008519-T2	-4.617470	NA	NA	NA		FALSE	FALSE

• Selected only genes that are potentially multicellularity related

mult_gene_table_ap2<-mult_gene_table %>% inner_join(ea_sig %>% filter(change=="apothecium") %>% select(target_id,b),by=c("TranscriptID"="target_id")) %>% arrange(Function)

mult_gene_table_ap2 %>% 
  kable(format = "html", col.names = colnames(mult_gene_table_ap2)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

TranscriptID	ID	Annotation_type	Description	Function	Function_type	b
XANPAGTX0501_001061-T1	IPR013149	IPR	Alcohol dehydrogenase-like, C-terminal	Acetyl-CoA production and metabolism	Fungal multicellularity	-1.603339
XANPAGTX0501_005597-T1	IPR032788	IPR	Glycogen debranching enzyme, central domain	Carbohydrate storage	Fungal multicellularity	-1.816345
XANPAGTX0501_000647-T1	IPR000571	IPR	Zinc finger, CCCH-type	Cell division, proliferation and growth	Fungal multicellularity	-1.940161
XANPAGTX0501_002213-T1	IPR001650	IPR	Helicase, C-terminal	Cell division, proliferation and growth	Fungal multicellularity	-1.688746
XANPAGTX0501_003775-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.770114
XANPAGTX0501_003775-T2	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.260916
XANPAGTX0501_004104-T1	IPR001214	IPR	SET domain	Cell division, proliferation and growth	Fungal multicellularity	-1.878122
XANPAGTX0501_004244-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.139706
XANPAGTX0501_004628-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.792127
XANPAGTX0501_003124-T1	IPR001461	IPR	Aspartic peptidase A1 family	Cell surface and cell wall proteins	Fungal multicellularity	-1.348872
XANPAGTX0501_005735-T1	IPR018824	IPR	Conidiation-specific protein 6	Cell surface and cell wall proteins	Fungal multicellularity	-2.102015
XANPAGTX0501_006327-T1	IPR018466	IPR	Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	Cell wall biosynthesis	Fungal multicellularity	-2.649849
XANPAGTX0501_000203-T1	IPR037176	IPR	Osmotin/thaumatin-like superfamily	Cell wall remodeling	Fungal multicellularity	-1.040557
XANPAGTX0501_001856-T1	IPR037176	IPR	Osmotin/thaumatin-like superfamily	Cell wall remodeling	Fungal multicellularity	-2.592477
XANPAGTX0501_007833-T1	IPR002889	IPR	Carbohydrate-binding WSC	Cell wall remodeling	Fungal multicellularity	-1.343993
XANPAGTX0501_009511-T1	GH16	CAZy	act on beta-1,4 or beta-1,3 glycosidic bonds in glucans and galactans	Cell wall remodeling	Fungal multicellularity	-1.533303
XANPAGTX0501_006767-T1	IPR017956	IPR	AT hook, DNA-binding motif	Chromatin regulation	Expression regulation	-1.001121
XANPAGTX0501_001064-T1	IPR021765	IPR	Mycotoxin biosynthesis protein UstYa-like	Defense	Fungal multicellularity	-1.621988
XANPAGTX0501_001643-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.910689
XANPAGTX0501_003212-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.757104
XANPAGTX0501_003213-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.804291
XANPAGTX0501_003264-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.264968
XANPAGTX0501_005550-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.294683
XANPAGTX0501_005620-T1	IPR017907	IPR	Zinc finger; RING-type; conserved site	Protein ubiquitination	Fungal multicellularity	-1.973360
XANPAGTX0501_006203-T1	IPR017907	IPR	Zinc finger; RING-type; conserved site	Protein ubiquitination	Fungal multicellularity	-1.386200
XANPAGTX0501_007869-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.472330
XANPAGTX0501_008856-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-2.160218
XANPAGTX0501_009316-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.697722
XANPAGTX0501_009738-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.170585
XANPAGTX0501_009942-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.494661
XANPAGTX0501_010380-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.404538
XANPAGTX0501_001648-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.214948
XANPAGTX0501_002098-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.334348
XANPAGTX0501_002123-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.312729
XANPAGTX0501_004217-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.766312
XANPAGTX0501_005177-T1	IPR001313	IPR	Pumilio RNA-binding repeat	RNA binding proteins	Expression regulation	-1.185673
XANPAGTX0501_002123-T1	IPR007855	IPR	RNA-dependent RNA polymerase, eukaryotic-type	RNA interference	Expression regulation	-1.312729
XANPAGTX0501_003775-T1	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.770114
XANPAGTX0501_003775-T2	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.260916
XANPAGTX0501_004244-T1	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.139706
XANPAGTX0501_000420-T1	IPR013087	IPR	Zinc finger C2H2-type	Transcription factors	Expression regulation	-1.518444
XANPAGTX0501_001955-T1	IPR036864	IPR	Zn(2)-C6 fungal-type DNA-binding domain superfamily	Transcription factors	Expression regulation	-1.387192
XANPAGTX0501_001958-T1	IPR001356	IPR	Homeobox domain	Transcription factors	Expression regulation	-1.998637
XANPAGTX0501_005620-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.973360
XANPAGTX0501_005640-T1	IPR013087	IPR	Zinc finger C2H2-type	Transcription factors	Expression regulation	-1.380432
XANPAGTX0501_006203-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.386200
XANPAGTX0501_006931-T1	IPR013087	IPR	Zinc finger C2H2-type	Transcription factors	Expression regulation	-1.072494
XANPAGTX0501_008005-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.251448
XANPAGTX0501_000281-T1	IPR036259	IPR	MFS transporter superfamily	Transporters	Fungal multicellularity	-1.282182
XANPAGTX0501_001825-T1	IPR036259	IPR	MFS transporter superfamily	Transporters	Fungal multicellularity	-1.402807
XANPAGTX0501_008594-T1	IPR036259	IPR	MFS transporter superfamily	Transporters	Fungal multicellularity	-1.040188
XANPAGTX0501_009737-T1	IPR036259	IPR	MFS transporter superfamily	Transporters	Fungal multicellularity	-1.540774

• Notably, many TFs and ubiquitin degradation proteins

• Also many transporters, which wasn’t the case in other lists here

• Enrichment analysis

enrich2<-clusterProfiler::enricher(ap_ea_genes$target_id,
    pAdjustMethod = "none",
    minGSSize = 1,
    maxGSSize = 2000,
    qvalueCutoff = 1,
    universe=ips_data$universe,
    TERM2GENE=ips_data$term2protein,
    TERM2NAME=ips_data$term2name)

enrich_pairwise2<-enrichplot::pairwise_termsim(enrich2)
enrichplot::emapplot(enrich_pairwise2)

Upregulated in edge

• Bootstrap of the two genes most overexpressed in the edge

plot_bootstrap(ea_so, 
               target_id = ea_sig$target_id[which.max(ea_sig$b)], 
               units = "est_counts", 
               color_by = "part")
plot_bootstrap(ea_so, 
               target_id = ea_sig$target_id[which.max(ea_sig$b[-which.max(ea_sig$b)])], 
               units = "est_counts", 
               color_by = "part")

• Of the 13 upregulated in edge, 3 have no functional annotations
• 6 are secreted

ed_ea_genes<-ea_sig %>% filter(change=="edge") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
ed_ea_genes %>% 
  kable(format = "html", col.names = colnames(ap_ea_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_010431-T1	1.523359	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001015-T1	1.339807	IPR001153 Barwin domain, IPR009009 RlpA-like protein, double-psi beta-barrel domain, IPR036908 RlpA-like domain superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_006189-T1	1.275509	IPR011701 Major facilitator superfamily, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002706-T1	1.129817	IPR002129 Pyridoxal phosphate-dependent decarboxylase, IPR015421 Pyridoxal phosphate-dependent transferase, major domain, IPR015424 Pyridoxal phosphate-dependent transferase	NA	NA		FALSE	FALSE
XANPAGTX0501_004034-T1	1.121819	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_010624-T1	1.121535	IPR023631 Amidase signature domain, IPR036928 Amidase signature (AS) superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_004943-T2	1.117316	IPR002020 Citrate synthase, IPR016142 Citrate synthase-like, large alpha subdomain, IPR016143 Citrate synthase-like, small alpha subdomain, IPR019810 Citrate synthase active site, IPR036969 Citrate synthase superfamily	NA	NA	K01647	FALSE	FALSE
XANPAGTX0501_006869-T1	1.099929	IPR008979 Galactose-binding-like domain superfamily, IPR017853 Glycoside hydrolase superfamily, IPR024655 Asl1-like, glycosyl hydrolase catalytic domain	GH128	NA		TRUE	FALSE
XANPAGTX0501_004633-T1	1.094695	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010430-T1	1.093667	IPR000542 Acyltransferase ChoActase/COT/CPT, IPR001227 Acyl transferase domain superfamily, IPR006162 Phosphopantetheine attachment site, IPR009081 Phosphopantetheine binding ACP domain, IPR011032 GroES-like superfamily, IPR013217 Methyltransferase type 12, IPR013968 Polyketide synthase, ketoreductase domain, IPR014030 Beta-ketoacyl synthase, N-terminal, IPR014031 Beta-ketoacyl synthase, C-terminal, IPR014043 Acyl transferase, IPR016035 Acyl transferase/acyl hydrolase/lysophospholipase, IPR016036 Malonyl-CoA ACP transacylase, ACP-binding, IPR016039 Thiolase-like, IPR020801 None, IPR020806 Polyketide synthase, phosphopantetheine-binding domain, IPR020807 Polyketide synthase, dehydratase domain, IPR020841 Polyketide synthase, beta-ketoacyl synthase domain, IPR020843 Polyketide synthase, enoylreductase domain, IPR029063 S-adenosyl-L-methionine-dependent methyltransferase superfamily, IPR032821 Polyketide synthase, C-terminal extension, IPR036291 NAD(P)-binding domain superfamily, IPR036736 ACP-like superfamily, IPR039551 Choline/carnitine acyltransferase domain, IPR042104 Polyketide synthase, dehydratase domain superfamily, IPR042231 Choline/Carnitine o-acyltransferase, domain 2, IPR042232 None	NA	NA	K12443	FALSE	TRUE
XANPAGTX0501_001458-T1	1.026449	IPR002889 Carbohydrate-binding WSC, IPR018535 Domain of unknown function DUF1996	NA	NA		TRUE	FALSE
XANPAGTX0501_009181-T1	1.024662	IPR008427 Extracellular membrane protein, CFEM domain	NA	NA		TRUE	TRUE
XANPAGTX0501_008853-T1	1.003145	IPR009799 EthD domain, IPR011008 Dimeric alpha-beta barrel	NA	NA		FALSE	FALSE

Parietin cluster is upregulated in the edge

• One of the proteins in the parietin BGC, an extra one

plot_bootstrap(ea_so, 
               target_id = "XANPAGTX0501_008853-T1", 
               units = "est_counts", 
               color_by = "part")

• The main PKS gene is just below the threshold (b = 0.92)

plot_bootstrap(ea_so, 
               target_id = "XANPAGTX0501_008852-T1", 
               units = "est_counts", 
               color_by = "part")

4.2. Power analysis: simple

• Do we have enough samples for this comparison?
• Looked at the distribution of corrected p-values (a.k.a. q-values). The graph looks good: close to uniform, with an increase close to 0. If our test was underpowered, we would see an increase of p-vlaues along 0->1, but this is not the case

ggplot(ea_table)+geom_histogram(aes(x=qval))

## `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.

## Warning: Removed 690798 rows containing non-finite values (`stat_bin()`).

• QQ-plot looks fine

library(MKpower)
qqunif(ea_table$qval, color.line = "orange")

5. How similar are gene lists between comparisons?

Upregulated in the edge

• Only one gene upregulated compared to the center, and it is also in the second list

library(ggVennDiagram)

## 
## Attaching package: 'ggVennDiagram'

## The following object is masked from 'package:tidyr':
## 
##     unite

venn_e<-list(compared_to_apothecia = ea_sig$target_id[ea_sig$change=="edge"],
               compared_to_center = ec_sig$target_id[ec_sig$b>0.9])
e<-ggVennDiagram(venn_e,label_size = 5,set_size=5)+labs(title = "Upregulated in edge")+theme(title=element_text(size=12))+scale_x_continuous(expand = expansion(mult = .4))
e

Upregulated in the center

• Compared to apothecia one is upregulated, and it’s not in the second list

venn_c<-list(compared_to_apothecia = ca_sig$target_id[ca_sig$change=="centre"],
               compared_to_edge = ec_sig$target_id[ec_sig$change=="centre"])

c<-ggVennDiagram(venn_c,label_size = 5,set_size=5)+labs(title = "Upregulated in center")+theme(title=element_text(size=12))+scale_x_continuous(expand = expansion(mult = .4))
c

Upregulated in the apothecia

• These lists are large, and the smaller is almost entirely overlapping with the bigger one

venn_a<-list(compared_to_center = ca_sig$target_id[ca_sig$change=="apothecium"],
               compared_to_edge = ea_sig$target_id[ea_sig$change=="apothecium"])

a<-ggVennDiagram(venn_a,label_size = 5,set_size=5)+labs(title = "Upregulated in apothecia")+theme(title=element_text(size=12))+scale_x_continuous(expand = expansion(mult = .4))
a

6. Comparing apothecia vs center+edge

• Since there was so little difference between edge and center, pooled them together

• 250 upregulated in apothecia, only 2 in thallus

kal_dirs2 <-kal_dirs2 %>% mutate(part2 = ifelse(part =="apothecia","apothecia","thallus") )
kal_dirs2$part2 <- as.factor(kal_dirs2$part2)
kal_dirs2$thallus<- as.factor(kal_dirs2$thallus)

rhdf5::h5closeAll()
a_so <- sleuth_prep(kal_dirs2,extra_bootstrap_summary = TRUE,filter_target_id = target_id,transformation_function = function(x) log2(x + 0.1))

## Warning in check_num_cores(num_cores): It appears that you are running Sleuth from within Rstudio.
## Because of concerns with forking processes from a GUI, 'num_cores' is being set to 1.
## If you wish to take advantage of multiple cores, please consider running sleuth from the command line.

## reading in kallisto results

## dropping unused factor levels

## .................
## normalizing est_counts
## A list of target IDs for filtering was found. Using this for filtering
## 11083 targets passed the filter
## normalizing tpm
## merging in metadata
## summarizing bootstraps
## .................

#model that includes only confounding variables
a_so <- sleuth_fit(a_so, ~thallus, 'reduced') 

## fitting measurement error models
## shrinkage estimation
## 1 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_000967-T1
## computing variance of betas

#model that includes only both the confounding variable and the variable of interest
a_so  <- sleuth_fit(a_so, ~ thallus + part2, 'full')

## fitting measurement error models
## shrinkage estimation
## 4 NA values were found during variance shrinkage estimation due to mean observation values outside of the range used for the LOESS fit.
## The LOESS fit will be repeated using exact computation of the fitted surface to extrapolate the missing values.
## These are the target ids with NA values: XANPAGTX0501_000967-T1, XANPAGTX0501_001127-T1, XANPAGTX0501_002409-T1, XANPAGTX0501_010086-T1
## computing variance of betas

#compare the models
a_so <- sleuth_lrt(a_so, 'reduced', 'full')
a_so <- sleuth_wt(a_so, 'part2thallus')

a_table <- sleuth_results(a_so, 'part2thallus')
a_sig <- a_table %>% tibble::as_tibble() %>% 
  filter( qval <= 0.05, grepl("GTX0501",target_id) ) %>% 
  arrange(desc(b)) %>% mutate(change = if_else(b > 1, "thallus", ifelse(b< -1, "apothecium", "low_logFC")))

table(a_sig$change)

## 
## apothecium  low_logFC    thallus 
##        250       2746          2

• raw and save PCA

library(RColorBrewer)
color_scheme<-c("apothecia"=brewer.pal(3,"Dark2")[1],
                "thallus_centre"=brewer.pal(3,"Dark2")[2], 
               "thallus_edge" =brewer.pal(3,"Dark2")[3] )

plot_pca(a_so, color_by = 'part', text_labels = TRUE,use_filtered=T)+
  theme_bw()+theme(text=element_text(size=7))+
  scale_color_manual(values=color_scheme)

ggsave('../results/tissue_pca.pdf',width = 4.5, height = 3)

Upregulated in thallus

plot_bootstrap(a_so, 
               target_id = a_sig$target_id[which.max(a_sig$b)], 
               units = "est_counts", 
               color_by = "part2")
plot_bootstrap(a_so, 
               target_id = a_sig$target_id[which.max(a_sig$b[-which.max(a_sig$b)])], 
               units = "est_counts", 
               color_by = "part2")

• XANPAGTX0501_009376-T1 was not DGE in the edge/apothecia and center/apothecia comparisons
  • is a ribosomal protein
• XANPAGTX0501_004633-T1 was upregulated in the edge in the edge/apothecia, and nearly upregulated in center/apothecia (b value = 0.977)
  • has no annotations, see above

a_genes<-a_sig %>% filter(b>0.9) %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
a_genes %>% 
  kable(format = "html", col.names = colnames(a_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_009376-T1	2.4961545	IPR043141 Ribosomal protein L10-like domain superfamily	NA	NA	K02864	FALSE	FALSE
XANPAGTX0501_004633-T1	1.0318823	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009913-T1	0.9922726	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_002706-T1	0.9523073	IPR002129 Pyridoxal phosphate-dependent decarboxylase, IPR015421 Pyridoxal phosphate-dependent transferase, major domain, IPR015424 Pyridoxal phosphate-dependent transferase	NA	NA		FALSE	FALSE

Upregulated in apothecia

• Of the 250 upregulated in apothecia, 171 have no functional annotations
• 24 are secreted

ap_a_genes<-a_sig %>% filter(change=="apothecium") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho)
ap_a_genes %>% 
  kable(format = "html", col.names = colnames(ap_a_genes)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho
XANPAGTX0501_007851-T1	-1.000996	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009787-T1	-1.001679	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000938-T1	-1.002687	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007833-T1	-1.003237	IPR002889 Carbohydrate-binding WSC, IPR018535 Domain of unknown function DUF1996	NA	NA		FALSE	FALSE
XANPAGTX0501_001498-T1	-1.006175	IPR012340 Nucleic acid-binding, OB-fold	NA	NA		FALSE	FALSE
XANPAGTX0501_005188-T1	-1.006230	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003775-T2	-1.015597	IPR003100 PAZ domain, IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032473 Protein argonaute, Mid domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K11593	FALSE	FALSE
XANPAGTX0501_007473-T1	-1.021407	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003790-T1	-1.024429	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006420-T1	-1.025603	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001955-T1	-1.028447	IPR001138 Zn(2)Cys(6) fungal-type DNA-binding domain, IPR001451 Hexapeptide repeat, IPR002110 Ankyrin repeat, IPR011004 Trimeric LpxA-like superfamily, IPR018357 Hexapeptide transferase, conserved site, IPR020683 Domain of unknown function DUF3447, IPR024688 Maltose/galactoside acetyltransferase, IPR036770 Ankyrin repeat-containing domain superfamily, IPR036864 Zn(2)-C6 fungal-type DNA-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_003033-T1	-1.030286	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002054-T1	-1.034049	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_003124-T1	-1.035285	IPR001461 Aspartic peptidase A1 family, IPR001969 Aspartic peptidase, active site, IPR021109 Aspartic peptidase domain superfamily, IPR033121 Peptidase family A1 domain, IPR034164 Pepsin-like domain	NA	A01A		TRUE	FALSE
XANPAGTX0501_002946-T1	-1.035401	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_002142-T1	-1.036917	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009581-T1	-1.037279	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004753-T1	-1.037567	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003765-T1	-1.038156	IPR000420 Yeast PIR protein repeat	NA	NA		TRUE	FALSE
XANPAGTX0501_004978-T1	-1.047426	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_004466-T1	-1.048483	IPR000490 Glycoside hydrolase family 17, IPR017853 Glycoside hydrolase superfamily, IPR018620 Ubiquitin 3 binding protein But2, C-terminal	NA	NA		TRUE	FALSE
XANPAGTX0501_006422-T1	-1.053965	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005317-T1	-1.054307	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_006975-T1	-1.054818	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000622-T1	-1.055181	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002098-T1	-1.056175	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_007365-T1	-1.056362	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004102-T1	-1.058681	IPR000420 Yeast PIR protein repeat	NA	NA	K26550	TRUE	FALSE
XANPAGTX0501_001825-T1	-1.063406	IPR003663 Sugar/inositol transporter, IPR005828 Major facilitator, sugar transporter-like, IPR005829 Sugar transporter, conserved site, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001223-T1	-1.064214	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005815-T1	-1.064277	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_000036-T1	-1.068838	IPR006640 SprT-like	NA	NA		FALSE	FALSE
XANPAGTX0501_002019-T1	-1.070414	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005619-T1	-1.070482	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009599-T1	-1.070929	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001964-T1	-1.071179	IPR001611 Leucine-rich repeat, IPR003591 Leucine-rich repeat, typical subtype, IPR019487 RAM signalling pathway, SOG2, IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_000888-T1	-1.071659	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009795-T2	-1.071736	IPR023631 Amidase signature domain, IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_008200-T1	-1.073076	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008888-T1	-1.075701	IPR007527 Zinc finger, SWIM-type	NA	NA		FALSE	TRUE
XANPAGTX0501_010001-T1	-1.075953	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010379-T1	-1.076421	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001965-T1	-1.078800	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010094-T1	-1.079269	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006459-T1	-1.089552	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002123-T1	-1.092697	IPR000504 RNA recognition motif domain, IPR007855 RNA-dependent RNA polymerase, eukaryotic-type, IPR035979 RNA-binding domain superfamily	NA	NA	K11699	FALSE	FALSE
XANPAGTX0501_009326-T1	-1.097552	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001948-T1	-1.099351	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009293-T1	-1.101651	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_007899-T1	-1.103335	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000420-T1	-1.104047	IPR013087 Zinc finger C2H2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_006246-T1	-1.104366	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006114-T1	-1.104804	IPR001680 WD40 repeat, IPR015943 WD40/YVTN repeat-like-containing domain superfamily, IPR036322 WD40-repeat-containing domain superfamily	NA	NA	K12782	FALSE	FALSE
XANPAGTX0501_006310-T1	-1.105045	IPR022137 Zinc finger protein, DUF3669 domain	NA	NA		FALSE	FALSE
XANPAGTX0501_001963-T1	-1.111833	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004008-T1	-1.117183	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002410-T1	-1.117363	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002963-T1	-1.118354	IPR000172 Glucose-methanol-choline oxidoreductase, N-terminal, IPR007867 Glucose-methanol-choline oxidoreductase, C-terminal, IPR012132 Glucose-methanol-choline oxidoreductase, IPR036188 FAD/NAD(P)-binding domain superfamily	AA3	NA	K00108	TRUE	FALSE
XANPAGTX0501_006909-T1	-1.120330	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010145-T1	-1.120565	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005584-T1	-1.124341	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002007-T1	-1.126910	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_005278-T1	-1.128439	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000872-T1	-1.128589	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001710-T1	-1.130646	IPR007577 Glycosyltransferase, DXD sugar-binding motif, IPR029044 Nucleotide-diphospho-sugar transferases	GT32	NA		FALSE	FALSE
XANPAGTX0501_007869-T1	-1.136332	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_009511-T1	-1.136478	IPR000757 Glycoside hydrolase family 16, IPR001002 Chitin-binding, type 1, IPR008264 Beta-glucanase, IPR013320 Concanavalin A-like lectin/glucanase domain superfamily, IPR036861 Endochitinase-like superfamily	GH16	NA	K01216	TRUE	FALSE
XANPAGTX0501_000691-T1	-1.140810	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010007-T1	-1.146366	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001820-T2	-1.147375	IPR021109 Aspartic peptidase domain superfamily, IPR033121 Peptidase family A1 domain	NA	NA		FALSE	FALSE
XANPAGTX0501_008863-T1	-1.149308	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010381-T1	-1.157248	IPR002293 Amino acid/polyamine transporter I	NA	NA	K03294	FALSE	FALSE
XANPAGTX0501_003361-T1	-1.157393	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_009666-T1	-1.163354	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000910-T1	-1.164861	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009942-T1	-1.164912	IPR001810 F-box domain, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_008874-T1	-1.168644	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005296-T1	-1.170842	IPR018750 Protein of unknown function DUF2306, membrane	NA	NA		FALSE	FALSE
XANPAGTX0501_004853-T1	-1.170891	IPR000719 Protein kinase domain, IPR000961 AGC-kinase, C-terminal, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_001512-T1	-1.171326	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006203-T1	-1.175049	IPR001841 Zinc finger, RING-type, IPR002867 IBR domain, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR017907 Zinc finger, RING-type, conserved site	NA	NA		FALSE	FALSE
XANPAGTX0501_001436-T1	-1.178216	IPR002048 EF-hand domain, IPR011992 EF-hand domain pair, IPR018247 EF-Hand 1, calcium-binding site	NA	NA		FALSE	FALSE
XANPAGTX0501_007750-T1	-1.181450	IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	FALSE
XANPAGTX0501_002820-T1	-1.183558	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010051-T1	-1.184568	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003247-T1	-1.184643	IPR001878 Zinc finger, CCHC-type, IPR025836 Zinc knuckle CX2CX4HX4C, IPR036875 Zinc finger, CCHC-type superfamily	NA	NA	K09250	FALSE	FALSE
XANPAGTX0501_009621-T1	-1.188204	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005620-T1	-1.188513	IPR001841 Zinc finger, RING-type, IPR004331 SPX domain, IPR011016 Zinc finger, RING-CH-type, IPR013083 Zinc finger, RING/FYVE/PHD-type, IPR017907 Zinc finger, RING-type, conserved site	NA	NA		FALSE	TRUE
XANPAGTX0501_009321-T1	-1.190228	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003448-T1	-1.191987	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_009141-T1	-1.192722	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009887-T1	-1.200786	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_000610-T1	-1.207426	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001643-T1	-1.215746	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_010380-T1	-1.223224	IPR000210 BTB/POZ domain, IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_007403-T1	-1.228285	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001082-T1	-1.232060	IPR004853 Sugar phosphate transporter domain	NA	NA	K26222	FALSE	FALSE
XANPAGTX0501_004416-T1	-1.234261	IPR000246 Peptidase T2, asparaginase 2, IPR029055 Nucleophile aminohydrolases, N-terminal, IPR037464 Threonine aspartase 1	NA	T02	K08657	FALSE	FALSE
XANPAGTX0501_001064-T1	-1.234644	IPR021765 Mycotoxin biosynthesis protein UstYa-like	NA	NA		FALSE	FALSE
XANPAGTX0501_001061-T1	-1.235309	IPR002328 Alcohol dehydrogenase, zinc-type, conserved site, IPR011032 GroES-like superfamily, IPR013149 Alcohol dehydrogenase-like, C-terminal, IPR013154 Alcohol dehydrogenase-like, N-terminal, IPR020843 Polyketide synthase, enoylreductase domain, IPR036291 NAD(P)-binding domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_004018-T2	-1.235695	IPR011009 Protein kinase-like domain superfamily, IPR016477 Fructosamine/Ketosamine-3-kinase	NA	NA		FALSE	TRUE
XANPAGTX0501_007231-T1	-1.240318	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009316-T1	-1.246011	IPR001810 F-box domain, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_003283-T1	-1.246856	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007244-T1	-1.249898	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008533-T1	-1.253515	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007343-T1	-1.255419	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001952-T1	-1.257146	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001950-T1	-1.259001	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002213-T1	-1.262177	IPR000629 ATP-dependent RNA helicase DEAD-box, conserved site, IPR001650 Helicase, C-terminal, IPR011545 DEAD/DEAH box helicase domain, IPR014001 Helicase superfamily 1/2, ATP-binding domain, IPR014014 RNA helicase, DEAD-box type, Q motif, IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	FALSE
XANPAGTX0501_001951-T1	-1.262779	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000604-T1	-1.264223	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010117-T1	-1.267941	IPR002110 Ankyrin repeat, IPR011009 Protein kinase-like domain superfamily, IPR016477 Fructosamine/Ketosamine-3-kinase, IPR020683 Domain of unknown function DUF3447, IPR036770 Ankyrin repeat-containing domain superfamily	NA	NA	K15523	FALSE	FALSE
XANPAGTX0501_000697-T1	-1.273117	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_008628-T1	-1.274944	IPR023346 Lysozyme-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_001956-T1	-1.276631	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000398-T1	-1.277824	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002057-T1	-1.282644	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001888-T1	-1.289043	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006449-T1	-1.291222	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008008-T1	-1.291576	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003282-T1	-1.293552	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008426-T1	-1.294283	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005613-T1	-1.294861	IPR003615 HNH nuclease	NA	NA		FALSE	TRUE
XANPAGTX0501_007464-T1	-1.298109	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007870-T1	-1.300798	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006245-T1	-1.301264	NA	NA	NA		TRUE	TRUE
XANPAGTX0501_008420-T1	-1.301707	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001947-T1	-1.303157	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006330-T1	-1.307915	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000892-T1	-1.309666	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010304-T1	-1.314210	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001362-T1	-1.314577	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004628-T1	-1.317480	IPR002156 Ribonuclease H domain, IPR012337 Ribonuclease H-like superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K03469	FALSE	FALSE
XANPAGTX0501_001330-T1	-1.320530	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005658-T1	-1.322458	IPR001153 Barwin domain, IPR009009 RlpA-like protein, double-psi beta-barrel domain, IPR036908 RlpA-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005402-T1	-1.324043	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_002165-T1	-1.325802	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008534-T1	-1.327391	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002170-T1	-1.328820	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009317-T1	-1.330469	IPR008979 Galactose-binding-like domain superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_005394-T1	-1.334492	IPR002575 Aminoglycoside phosphotransferase, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_005631-T1	-1.336475	IPR023631 Amidase signature domain, IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002103-T1	-1.336659	IPR036910 High mobility group box domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_008856-T1	-1.339511	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_000681-T1	-1.344215	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002185-T1	-1.352061	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003775-T1	-1.352148	IPR003100 PAZ domain, IPR003165 Piwi domain, IPR012337 Ribonuclease H-like superfamily, IPR014811 Argonaute, linker 1 domain, IPR032472 Argonaute linker 2 domain, IPR032473 Protein argonaute, Mid domain, IPR032474 Protein argonaute, N-terminal, IPR036085 PAZ domain superfamily, IPR036397 Ribonuclease H superfamily	NA	NA	K11593	FALSE	FALSE
XANPAGTX0501_004580-T1	-1.355977	IPR007292 Nuclear fusion protein Kar5	NA	NA		FALSE	FALSE
XANPAGTX0501_001010-T1	-1.356734	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002104-T1	-1.360844	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004494-T1	-1.362189	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001859-T1	-1.367755	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000025-T1	-1.368135	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_005597-T1	-1.371604	IPR006421 Glycogen debranching enzyme, metazoa, IPR008928 Six-hairpin glycosidase superfamily, IPR012341 Six-hairpin glycosidase-like superfamily, IPR017853 Glycoside hydrolase superfamily, IPR029436 Eukaryotic glycogen debranching enzyme, N-terminal domain, IPR032788 Glycogen debranching enzyme, central domain, IPR032790 Glycogen debranching enzyme, C-terminal, IPR032792 Glycogen debranching enzyme, glucanotransferase domain	GH133	NA	K01196	FALSE	FALSE
XANPAGTX0501_004950-T1	-1.372326	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005048-T1	-1.374486	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004217-T1	-1.376777	IPR000504 RNA recognition motif domain, IPR012677 Nucleotide-binding alpha-beta plait domain superfamily, IPR035979 RNA-binding domain superfamily	NA	NA	K24990	FALSE	FALSE
XANPAGTX0501_000005-T1	-1.378844	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003212-T1	-1.384927	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_002763-T1	-1.389644	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002163-T1	-1.392989	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003213-T1	-1.399730	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE
XANPAGTX0501_004493-T1	-1.400770	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002166-T1	-1.400943	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010280-T1	-1.404057	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006016-T1	-1.404604	IPR008928 Six-hairpin glycosidase superfamily, IPR031335 Glycosyl hydrolase family 63, C-terminal	GH63	NA		FALSE	FALSE
XANPAGTX0501_001333-T1	-1.411305	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_004104-T1	-1.419256	IPR001214 SET domain, IPR011990 Tetratricopeptide-like helical domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_007067-T1	-1.420720	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_007300-T1	-1.423032	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004414-T1	-1.423458	IPR027417 P-loop containing nucleoside triphosphate hydrolase	NA	NA		FALSE	TRUE
XANPAGTX0501_001605-T1	-1.425833	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006255-T1	-1.434751	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000647-T1	-1.440550	IPR000571 Zinc finger, CCCH-type	NA	NA		FALSE	FALSE
XANPAGTX0501_005615-T1	-1.440903	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006974-T1	-1.441975	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006957-T1	-1.443602	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006910-T1	-1.458293	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001612-T1	-1.458506	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007871-T1	-1.463016	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_009582-T1	-1.469666	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009615-T1	-1.478640	IPR035940 CAP superfamily	NA	NA		TRUE	FALSE
XANPAGTX0501_001953-T1	-1.483265	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009139-T1	-1.494375	IPR001876 Zinc finger, RanBP2-type	NA	NA		FALSE	FALSE
XANPAGTX0501_005630-T1	-1.497428	IPR036928 Amidase signature (AS) superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_004891-T1	-1.501545	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002522-T1	-1.504550	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005612-T1	-1.506602	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000893-T1	-1.508341	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005061-T1	-1.508665	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_007873-T1	-1.510868	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_001958-T1	-1.511188	IPR001356 Homeobox domain, IPR009057 Homeobox-like domain superfamily, IPR017970 Homeobox, conserved site	NA	NA		FALSE	FALSE
XANPAGTX0501_000194-T1	-1.511338	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR013780 Glycosyl hydrolase, all-beta, IPR017853 Glycoside hydrolase superfamily	GH13	NA	K01176	FALSE	FALSE
XANPAGTX0501_003572-T1	-1.519271	IPR001087 GDSL lipase/esterase, IPR036514 SGNH hydrolase superfamily	CE16	NA		TRUE	FALSE
XANPAGTX0501_002183-T1	-1.522262	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005279-T1	-1.523896	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008041-T1	-1.527220	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_005614-T1	-1.527719	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001538-T1	-1.528884	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001954-T1	-1.534783	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001414-T1	-1.537299	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001910-T1	-1.539771	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010303-T1	-1.542026	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004520-T1	-1.559567	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007262-T1	-1.569541	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_010203-T1	-1.569541	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_008090-T1	-1.570035	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009318-T1	-1.577677	IPR011333 SKP1/BTB/POZ domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002164-T1	-1.582652	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002184-T1	-1.584723	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005735-T1	-1.620055	IPR018824 Conidiation-specific protein 6	NA	NA		FALSE	FALSE
XANPAGTX0501_004521-T1	-1.625040	IPR008271 Serine/threonine-protein kinase, active site, IPR011009 Protein kinase-like domain superfamily	NA	NA		FALSE	FALSE
XANPAGTX0501_002172-T1	-1.626040	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001967-T1	-1.629031	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008009-T1	-1.631945	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009605-T1	-1.639551	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009619-T1	-1.640066	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001611-T1	-1.646921	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005766-T1	-1.650350	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_002182-T1	-1.670548	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008782-T1	-1.681484	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009786-T1	-1.691780	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001962-T1	-1.692967	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007872-T1	-1.694556	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_006253-T1	-1.695976	IPR014849 EKC/KEOPS complex, subunit Gon7	NA	NA		FALSE	FALSE
XANPAGTX0501_008331-T1	-1.707064	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003209-T1	-1.709229	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_000193-T1	-1.729825	IPR006047 Glycosyl hydrolase, family 13, catalytic domain, IPR017853 Glycoside hydrolase superfamily	GH13	NA		FALSE	FALSE
XANPAGTX0501_001960-T1	-1.733269	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_006951-T1	-1.742660	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008007-T1	-1.745209	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_010596-T1	-1.748698	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_005814-T1	-1.760286	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008284-T1	-1.771059	IPR029044 Nucleotide-diphospho-sugar transferases	GT2_Glyco_tranf_2	NA		FALSE	FALSE
XANPAGTX0501_009926-T1	-1.775143	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_009602-T1	-1.777131	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001063-T1	-1.783787	NA	NA	NA		TRUE	FALSE
XANPAGTX0501_007397-T1	-1.849425	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_007900-T1	-1.867603	NA	NA	NA		FALSE	TRUE
XANPAGTX0501_000378-T1	-1.880561	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_004966-T1	-1.889077	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001856-T1	-1.894636	IPR037176 Osmotin/thaumatin-like superfamily	NA	NA		TRUE	TRUE
XANPAGTX0501_006327-T1	-1.922253	IPR018466 Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	NA	NA		TRUE	FALSE
XANPAGTX0501_002506-T1	-1.931043	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_003211-T1	-2.074356	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_008109-T1	-2.159140	IPR004143 Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL), catalytic domain	NA	NA	K03800	FALSE	FALSE
XANPAGTX0501_004085-T1	-2.298435	IPR000420 Yeast PIR protein repeat	NA	NA	K26550	TRUE	FALSE
XANPAGTX0501_008519-T2	-2.955922	NA	NA	NA		FALSE	FALSE
XANPAGTX0501_001156-T3	-3.105399	IPR003822 Paired amphipathic helix, IPR013194 Histone deacetylase interacting domain, IPR031693 Sin3, C-terminal, IPR036600 Paired amphipathic helix superfamily	NA	NA	K11644	FALSE	FALSE

• Selected only genes that are potentially multicellularity related

mult_gene_table_ap3<-mult_gene_table %>% inner_join(a_sig %>% filter(change=="apothecium") %>% select(target_id,b),by=c("TranscriptID"="target_id")) %>% arrange(Function)

mult_gene_table_ap3 %>% 
  kable(format = "html", col.names = colnames(mult_gene_table_ap3)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "600px")

TranscriptID	ID	Annotation_type	Description	Function	Function_type	b
XANPAGTX0501_001061-T1	IPR013149	IPR	Alcohol dehydrogenase-like, C-terminal	Acetyl-CoA production and metabolism	Fungal multicellularity	-1.235309
XANPAGTX0501_005597-T1	IPR032788	IPR	Glycogen debranching enzyme, central domain	Carbohydrate storage	Fungal multicellularity	-1.371604
XANPAGTX0501_000647-T1	IPR000571	IPR	Zinc finger, CCCH-type	Cell division, proliferation and growth	Fungal multicellularity	-1.440550
XANPAGTX0501_002213-T1	IPR001650	IPR	Helicase, C-terminal	Cell division, proliferation and growth	Fungal multicellularity	-1.262177
XANPAGTX0501_003775-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.352148
XANPAGTX0501_003775-T2	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.015597
XANPAGTX0501_004104-T1	IPR001214	IPR	SET domain	Cell division, proliferation and growth	Fungal multicellularity	-1.419256
XANPAGTX0501_004628-T1	IPR012337	IPR	Ribonuclease H-like superfamily	Cell division, proliferation and growth	Fungal multicellularity	-1.317480
XANPAGTX0501_003124-T1	IPR001461	IPR	Aspartic peptidase A1 family	Cell surface and cell wall proteins	Fungal multicellularity	-1.035285
XANPAGTX0501_005735-T1	IPR018824	IPR	Conidiation-specific protein 6	Cell surface and cell wall proteins	Fungal multicellularity	-1.620055
XANPAGTX0501_006327-T1	IPR018466	IPR	Yeast cell wall synthesis Kre9/Knh1-like, N-terminal	Cell wall biosynthesis	Fungal multicellularity	-1.922253
XANPAGTX0501_001856-T1	IPR037176	IPR	Osmotin/thaumatin-like superfamily	Cell wall remodeling	Fungal multicellularity	-1.894636
XANPAGTX0501_007833-T1	IPR002889	IPR	Carbohydrate-binding WSC	Cell wall remodeling	Fungal multicellularity	-1.003237
XANPAGTX0501_009511-T1	GH16	CAZy	act on beta-1,4 or beta-1,3 glycosidic bonds in glucans and galactans	Cell wall remodeling	Fungal multicellularity	-1.136478
XANPAGTX0501_001064-T1	IPR021765	IPR	Mycotoxin biosynthesis protein UstYa-like	Defense	Fungal multicellularity	-1.234644
XANPAGTX0501_001643-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.215746
XANPAGTX0501_003212-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.384927
XANPAGTX0501_003213-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.399730
XANPAGTX0501_005620-T1	IPR017907	IPR	Zinc finger; RING-type; conserved site	Protein ubiquitination	Fungal multicellularity	-1.188513
XANPAGTX0501_005815-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.064277
XANPAGTX0501_006203-T1	IPR017907	IPR	Zinc finger; RING-type; conserved site	Protein ubiquitination	Fungal multicellularity	-1.175049
XANPAGTX0501_007869-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.136332
XANPAGTX0501_008856-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.339511
XANPAGTX0501_009316-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.246011
XANPAGTX0501_009942-T1	IPR001810	IPR	F-box domain	Protein ubiquitination	Fungal multicellularity	-1.164912
XANPAGTX0501_010380-T1	IPR000210	IPR	BTB/POZ domain	Protein ubiquitination	Fungal multicellularity	-1.223224
XANPAGTX0501_002098-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.056175
XANPAGTX0501_002123-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.092697
XANPAGTX0501_004217-T1	IPR000504	IPR	RNA recognition motif domain	RNA binding proteins	Expression regulation	-1.376777
XANPAGTX0501_002123-T1	IPR007855	IPR	RNA-dependent RNA polymerase, eukaryotic-type	RNA interference	Expression regulation	-1.092697
XANPAGTX0501_003775-T1	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.352148
XANPAGTX0501_003775-T2	IPR032474	IPR	Protein argonaute, N-terminal	RNA interference	Expression regulation	-1.015597
XANPAGTX0501_000420-T1	IPR013087	IPR	Zinc finger C2H2-type	Transcription factors	Expression regulation	-1.104047
XANPAGTX0501_001955-T1	IPR036864	IPR	Zn(2)-C6 fungal-type DNA-binding domain superfamily	Transcription factors	Expression regulation	-1.028447
XANPAGTX0501_001958-T1	IPR001356	IPR	Homeobox domain	Transcription factors	Expression regulation	-1.511188
XANPAGTX0501_005620-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.188513
XANPAGTX0501_006203-T1	IPR013083	IPR	Zinc finger, RING/FYVE/PHD-type	Transcription factors	Expression regulation	-1.175049
XANPAGTX0501_001825-T1	IPR036259	IPR	MFS transporter superfamily	Transporters	Fungal multicellularity	-1.063406

• Enrichment analysis

###enrichment analysis
enrich<-clusterProfiler::enricher(ap_a_genes$target_id,
    pAdjustMethod = "none",
    minGSSize = 1,
    maxGSSize = 2000,
    qvalueCutoff = 1,
    universe=ips_data$universe,
    TERM2GENE=ips_data$term2protein,
    TERM2NAME=ips_data$term2name)

enrich_pairwise<-enrichplot::pairwise_termsim(enrich)
enrichplot::emapplot(enrich_pairwise)

• Save

pdf(file="../results/apothecia_upr.pdf",width=6,height=5)
enrichplot::emapplot(enrich_pairwise,cex_label_category=0.4,cex_line=0.25,
                     shadowtext=F,cex_pie2axis=0.1,repel=T)
dev.off()

## quartz_off_screen 
##                 2

• This new set includes almost all of the genes from the center/apothecia comparison. It also includes 59% of the genes from the edge/apothecia comparison. It also includes 12 genes unique to this set

venn_a2<-list(compared_to_center = ca_sig$target_id[ca_sig$change=="apothecium"],
               compared_to_edge = ea_sig$target_id[ea_sig$change=="apothecium"],
              compared_to_center_edge_pooled=a_sig$target_id[a_sig$change=="apothecium"])

a2<-ggVennDiagram(venn_a2,label_size = 5,set_size=5)+labs(title = "Upregulated in apothecia")+theme(title=element_text(size=12))+scale_x_continuous(expand = expansion(mult = .4))
a2

7. Visualize MAT expression

• Standartd heatmap - not very clear

library(ComplexHeatmap)
library(viridis)
#make a matrix
tabd_df_a <- a_so$obs_norm[a_so$obs_norm$target_id %in% target_id,]
tabd_df_a <- dplyr::select(tabd_df_a, target_id, sample, 
            tpm)
tabd_df_a <- reshape2::dcast(tabd_df_a, target_id ~ sample, 
            value.var = "tpm") 
rownames(tabd_df_a) <- tabd_df_a$target_id
tabd_df_a$target_id <- NULL
trans_mat_a <- as.matrix(log(tabd_df_a + 1))

#make a top annotation
#define column annotations
s2c_a <- a_so$sample_to_covariates
s2c_a<-data.frame("sample"= colnames(tabd_df_a)) %>% left_join(s2c_a)
rownames(s2c_a)<-s2c_a$sample
s2c_a<-s2c_a %>% select(part)
ta_colors_a = c("apothecia" = "#619CFF", "thallus_edge" = "#F8766D","thallus_centre"="green")
ta_a = HeatmapAnnotation(df=s2c_a,col = list(condition = ta_colors_a))

#filter to include only MAT locus
filt_mat_MAT<-subset(trans_mat_a, rownames(trans_mat_a) %in% c("XANPAGTX0501_002103-T1","XANPAGTX0501_002104-T1"))
HM_mat = Heatmap(filt_mat_MAT, show_row_names = T, show_column_names = T,cluster_rows = T, row_title_rot = 0, col=viridis(100), heatmap_legend_param = list(title = "Expression: log(TPM)"), top_annotation = ta_a)

pdf(file="../results/MAT_heatmap.pdf",width=10,height=2)
draw(HM_mat)
dev.off()

## quartz_off_screen 
##                 2

HM_mat

* lolipop plot arranged by specimen

df<-data.frame(filt_mat_MAT) %>% mutate(target_id=rownames(filt_mat_MAT)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)
write.table(df, file="../analysis_and_temp_files/09_dge_architecture/mat_expression.txt", quote=F, sep='\t', row.names=F)

#only include thalli that a. have apothecia sample and b. have at least two samples
incl<-a_so$sample_to_covariates %>% mutate(if_apothecia=ifelse(part=="apothecia",T,F)) %>% 
  group_by(thallus) %>% summarize(n=n(),n_ap=sum(if_apothecia)) %>% filter(n>1,n_ap>0)
  
gg<-ggplot(df %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45))+
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6),legend.position="bottom")

ggsave('../results/MAT_lopipop.pdf',gg, width = 4, height = 3)
gg

Investigate the most apothecia-upregulated proteins

• XANPAGTX0501_001156-T3 is one variant of Sin3 transcriptional regulator
• Only one is upregulated

filt_mat_sin3<-subset(trans_mat_a, rownames(trans_mat_a) %in% c("XANPAGTX0501_001156-T1","XANPAGTX0501_001156-T2","XANPAGTX0501_001156-T3"))
HM_sin3 = Heatmap(filt_mat_sin3, show_row_names = T, show_column_names = T,cluster_rows = T, row_title_rot = 0, col=viridis(100), heatmap_legend_param = list(title = "Expression: log(TPM)"), top_annotation = ta_a)
draw(HM_sin3)

df_sin3<-data.frame(filt_mat_sin3) %>% mutate(target_id=rownames(filt_mat_sin3)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)

## Joining with `by = join_by(sample)`

ggplot(df_sin3 %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  scale_color_manual( values = color_scheme)+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45))

• This is very interesting, but also inconsistent. Looked at the bam file but it didn’t clarify much

• Visualize several key gene for the suppl. figure

  • XANPAGTX0501_004521-T1: Serine/threonine-protein kinase
  • XANPAGTX0501_001958-T1: Homeobox domain
  • XANPAGTX0501_003775-T1,XANPAGTX0501_004244-T1: argonaute
  • XANPAGTX0501_002123-T1: RNA-dependent RNA polymerase
  • XANPAGTX0501_004580-T1: Nuclear fusion protein Kar5
  • XANPAGTX0501_005735-T1: Conidiation-specific protein 6
  • XANPAGTX0501_004217-T1, XANPAGTX0501_002123-T1, XANPAGTX0501_002098-T1: RNA-binding domain
  • XANPAGTX0501_003213-T1, XANPAGTX0501_008856-T1, XANPAGTX0501_009316-T1, XANPAGTX0501_009942-T1, XANPAGTX0501_003212-T1, XANPAGTX0501_001643-T1: F-box
  • XANPAGTX0501_005815-T1,XANPAGTX0501_007869-T1,XANPAGTX0501_010380-T1: POZ

library(patchwork)
ap_genes<-c("XANPAGTX0501_004521-T1","XANPAGTX0501_001958-T1","XANPAGTX0501_004580-T1","XANPAGTX0501_005735-T1","XANPAGTX0501_002123-T1","XANPAGTX0501_003775-T1","XANPAGTX0501_004244-T1","XANPAGTX0501_004217-T1", "XANPAGTX0501_002098-T1")

filt_mat_ap<-subset(trans_mat_a, rownames(trans_mat_a) %in% ap_genes)
df_ap<-data.frame(filt_mat_ap) %>% mutate(target_id=rownames(filt_mat_ap)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)
df_ap$target_id<-factor(df_ap$target_id,levels=c("XANPAGTX0501_004521-T1","XANPAGTX0501_001958-T1","XANPAGTX0501_004580-T1","XANPAGTX0501_005735-T1","XANPAGTX0501_002123-T1","XANPAGTX0501_003775-T1","XANPAGTX0501_004244-T1",
    "XANPAGTX0501_004217-T1", "XANPAGTX0501_002098-T1") )

gg_ap<-ggplot(df_ap %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45))+
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6))

ap_genes2<-c("XANPAGTX0501_003213-T1", "XANPAGTX0501_008856-T1", "XANPAGTX0501_009316-T1","XANPAGTX0501_009942-T1", "XANPAGTX0501_003212-T1", "XANPAGTX0501_001643-T1","XANPAGTX0501_005815-T1","XANPAGTX0501_007869-T1",
                    "XANPAGTX0501_010380-T1")

filt_mat_ap2<-subset(trans_mat_a, rownames(trans_mat_a) %in% ap_genes2)
df_ap2<-data.frame(filt_mat_ap2) %>% mutate(target_id=rownames(filt_mat_ap2)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)
df_ap2$target_id<-factor(df_ap2$target_id,levels=c("XANPAGTX0501_003213-T1", "XANPAGTX0501_008856-T1", "XANPAGTX0501_009316-T1","XANPAGTX0501_009942-T1", "XANPAGTX0501_003212-T1", "XANPAGTX0501_001643-T1","XANPAGTX0501_005815-T1","XANPAGTX0501_007869-T1",
                    "XANPAGTX0501_010380-T1") )

gg_ap2<-ggplot(df_ap2 %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) +
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6))

gg_ap_all<-gg_ap+gg_ap2+ plot_layout(axis_titles = "collect",guides = 'collect')
ggsave('../results/apothecia_dge_lopipop.pdf',gg_ap_all, width = 8, height = 10)

• Visualize a couple most interesting for the main figure on apothecia

#get the matrix for all samples, including culture
tabd_df<-read.delim2("../analysis_and_temp_files/08_dge_culture_lichen/norm_counts_sleuth.txt")
rownames(tabd_df) <- tabd_df$target_id
tabd_df$target_id <- NULL
tabd_df <- mutate_all(tabd_df, function(x) as.numeric(as.character(x)))
trans_mat_08 <- as.matrix(log(tabd_df + 1))


selected_genes<-c("XANPAGTX0501_008856-T1","XANPAGTX0501_002123-T1")
#graph for the lichen samples
filt_mat_selected_thalli<-subset(trans_mat_a, rownames(trans_mat_a) %in% selected_genes)

df_thalli<-data.frame(filt_mat_selected_thalli) %>% mutate(target_id=rownames(filt_mat_selected_thalli)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)

## Joining with `by = join_by(sample)`

df_thalli$target_id<-factor(df_thalli$target_id,levels=c("XANPAGTX0501_008856-T1","XANPAGTX0501_002123-T1"))

gg_selected_thalli<-ggplot(df_thalli %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) +
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6),legend.position = "bottom",
          axis.text.x = element_blank(),axis.ticks.x = element_blank())

#graph for the culture
filt_mat_selected_culture<-subset(trans_mat_08, rownames(trans_mat_08) %in% selected_genes)
df_culture<-data.frame(filt_mat_selected_culture) %>% mutate(target_id=rownames(filt_mat_selected_culture)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>%
  filter(!(sample %in% colnames(filt_mat_selected_thalli)))
df_culture$target_id<-factor(df_culture$target_id,levels=c("XANPAGTX0501_008856-T1","XANPAGTX0501_002123-T1"))

gg_selected_culture<-ggplot(df_culture, aes(y=expression,x=sample))+
  geom_segment( aes(x=sample, xend=sample, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~.)+
  xlab("")+ylab("")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) + theme(text=element_text(size=6),axis.text = element_blank(),
                              axis.ticks = element_blank(),
                              axis.title.y = element_blank() )

ggsel<-gg_selected_thalli + gg_selected_culture + plot_layout(widths = c(0.6,0.4),nrow = 1) & scale_y_continuous(limits = c(0, 2.5))

#had to make one of them separately baceaus of the axes issue
selected_genes<-c("XANPAGTX0501_003775-T1")

#graph for the lichen samples
filt_mat_selected_thalli<-subset(trans_mat_a, rownames(trans_mat_a) %in% selected_genes)

df_thalli<-data.frame(filt_mat_selected_thalli) %>% mutate(target_id=rownames(filt_mat_selected_thalli)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)

## Joining with `by = join_by(sample)`

#df_thalli$target_id<-factor(df_selected$target_id,levels=c())

gg_selected_thalli<-ggplot(df_thalli %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) +
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6),legend.position = "bottom",
      strip.background.x = element_blank(),strip.text.x = element_blank())

#graph for the culture
filt_mat_selected_culture<-subset(trans_mat_08, rownames(trans_mat_08) %in% selected_genes)
df_culture<-data.frame(filt_mat_selected_culture) %>% mutate(target_id=rownames(filt_mat_selected_culture)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>%
  filter(!(sample %in% colnames(filt_mat_selected_thalli)))
#df_thalli$target_id<-factor(df_selected$target_id,levels=c())

gg_selected_culture<-ggplot(df_culture, aes(y=expression,x=sample))+
  geom_segment( aes(x=sample, xend=sample, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~.)+
  xlab("")+ylab("")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) + theme(text=element_text(size=6),axis.text.y = element_blank(),
                              axis.ticks.y = element_blank(),
                              axis.title.y = element_blank() )

ggsel2<-gg_selected_thalli + gg_selected_culture + plot_layout(widths = c(0.6,0.4),nrow = 1) & scale_y_continuous(limits = c(0, 5))

sel_final<-ggsel/plot_spacer()/ggsel2+ plot_layout(axis_titles = "collect",guides = "collect",heights = c(0.66,-0.155,0.34)) &theme(legend.position = "bottom")
ggsave('../results/selected_apothecia_dge_lopipop.pdf',sel_final, width = 7, height = 4)

sel_final

VIsualize interesting edge-upregulated genes

#get the matrix for all samples, including culture

selected_genes<-c("XANPAGTX0501_009376-T1","XANPAGTX0501_008852-T1")

#graph for the lichen samples
filt_mat_selected_thalli<-subset(trans_mat_a, rownames(trans_mat_a) %in% selected_genes)

df_thalli<-data.frame(filt_mat_selected_thalli) %>% mutate(target_id=rownames(filt_mat_selected_thalli)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)

## Joining with `by = join_by(sample)`

gg_selected_thalli<-ggplot(df_thalli %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) +
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6),legend.position = "bottom",
              strip.background.x = element_blank(),strip.text.x = element_blank())

#graph for the culture
filt_mat_selected_culture<-subset(trans_mat_08, rownames(trans_mat_08) %in% selected_genes)
df_culture<-data.frame(filt_mat_selected_culture) %>% mutate(target_id=rownames(filt_mat_selected_culture)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>%
  filter(!(sample %in% colnames(filt_mat_selected_thalli)))


gg_selected_culture<-ggplot(df_culture, aes(y=expression,x=sample))+
  geom_segment( aes(x=sample, xend=sample, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~.)+
  xlab("")+ylab("")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) + theme(text=element_text(size=6),axis.text.y = element_blank(),
                              axis.ticks.y = element_blank(),
                              axis.title.y = element_blank() )

ggsel<-gg_selected_thalli + gg_selected_culture + plot_layout(widths = c(0.6,0.4),nrow = 1) & scale_y_continuous(limits = c(0, 5))

#had to make one of them separately baceaus of the axes issue
selected_genes<-c("XANPAGTX0501_001015-T1")
selected_genes<-c("XANPAGTX0501_001643-T1")


#graph for the lichen samples
filt_mat_selected_thalli<-subset(trans_mat_a, rownames(trans_mat_a) %in% selected_genes)

df_thalli<-data.frame(filt_mat_selected_thalli) %>% mutate(target_id=rownames(filt_mat_selected_thalli)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>% left_join(a_so$sample_to_covariates)

## Joining with `by = join_by(sample)`

gg_selected_thalli<-ggplot(df_thalli %>% filter(thallus %in% incl$thallus), aes(color=part,y=expression,x=part))+
  geom_segment( aes(x=part, xend=part, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~thallus)+
  xlab("")+ylab("Expression: log(TPM)")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) +
  scale_color_manual( values = color_scheme)+
  theme(text=element_text(size=6),legend.position = "bottom",
        axis.text.x = element_blank(),
                              axis.ticks.x = element_blank(),
                              axis.title.x = element_blank() )

#graph for the culture
filt_mat_selected_culture<-subset(trans_mat_08, rownames(trans_mat_08) %in% selected_genes)
df_culture<-data.frame(filt_mat_selected_culture) %>% mutate(target_id=rownames(filt_mat_selected_culture)) %>%
  pivot_longer(-target_id,names_to="sample",values_to="expression") %>%
  filter(!(sample %in% colnames(filt_mat_selected_thalli)))
#df_thalli$target_id<-factor(df_selected$target_id,levels=c())

gg_selected_culture<-ggplot(df_culture, aes(y=expression,x=sample))+
  geom_segment( aes(x=sample, xend=sample, y=0, yend=expression))+
  geom_point(stat = "identity",position=position_dodge(width = .5))+
  facet_grid(target_id~.)+
  xlab("")+ylab("")+
  theme_bw()+ scale_x_discrete(guide = guide_axis(angle = 45)) + theme(text=element_text(size=6),axis.text = element_blank(),
                              axis.ticks = element_blank(),
                              axis.title = element_blank() )

ggsel2<-gg_selected_thalli + gg_selected_culture + plot_layout(widths = c(0.6,0.4),nrow = 1) & scale_y_continuous(limits = c(0, 7))

sel_final<-ggsel2/plot_spacer()/ggsel+ plot_layout(axis_titles = "collect",guides = "collect",heights = c(0.34,-0.105,0.66)) &theme(legend.position = "bottom")
ggsave('../results/selected_edge_dge_lopipop.pdf',sel_final, width = 7, height = 4)

sel_final

7. Compiling DGE table

ec_sig$Comparison<-"Edge / Center"
ea_sig$Comparison<-"Edge / Apothecia"
ca_sig$Comparison<-"Center / Apothecia"
a_sig$Comparison<-"Apothecia / Vegetative (edge + center)"

compiled<-rbind(ec_sig,ea_sig,ca_sig,a_sig) %>% filter(change!="low_logFC") %>%
  left_join(funannot2,by=c("target_id"="TranscriptID")) %>%
  select(Comparison,change,target_id,b,InterPro_new,CAZyme_new,Protease_new,KO,secreted_consensus,lichen_ortho) %>% arrange(change) %>% arrange(Comparison)
write.table(compiled, file="../results/tissue_dge.txt", quote=F, sep='\t', row.names=F)

The gene that are upregulated in the center compared to the edge, are they also apothecia-upregulated?

• All of them are at least in one apothecia-upregulated list

apothecia_up<-compiled$target_id[compiled$change=="apothecium"]
center_up<-ec_sig$target_id[ec_sig$change=="centre"]

ec_sig %>% filter(change=="centre") %>% 
  mutate(apothecia_upregulated = ifelse(target_id %in%apothecia_up,T,F)) %>%
  select(target_id,apothecia_upregulated,b)

## # A tibble: 9 × 3
##   target_id              apothecia_upregulated     b
##   <chr>                  <lgl>                 <dbl>
## 1 XANPAGTX0501_005615-T1 TRUE                  -1.01
## 2 XANPAGTX0501_005614-T1 TRUE                  -1.08
## 3 XANPAGTX0501_008007-T1 TRUE                  -1.16
## 4 XANPAGTX0501_002184-T1 TRUE                  -1.17
## 5 XANPAGTX0501_001949-T1 TRUE                  -1.19
## 6 XANPAGTX0501_002506-T1 TRUE                  -1.19
## 7 XANPAGTX0501_008856-T1 TRUE                  -1.28
## 8 XANPAGTX0501_009886-T1 TRUE                  -1.29
## 9 XANPAGTX0501_009927-T1 TRUE                  -1.62

• All of them were in the apothecia/edge list

compiled %>% filter(change=="apothecium", target_id %in% center_up) %>%
   select(target_id,Comparison) %>% mutate(presence=1) %>%
  pivot_wider(names_from = Comparison,values_from = presence, values_fill=0)

## # A tibble: 9 × 4
##   target_id       Apothecia / Vegetati…¹ `Center / Apothecia` `Edge / Apothecia`
##   <chr>                            <dbl>                <dbl>              <dbl>
## 1 XANPAGTX0501_0…                      1                    0                  1
## 2 XANPAGTX0501_0…                      1                    1                  1
## 3 XANPAGTX0501_0…                      1                    0                  1
## 4 XANPAGTX0501_0…                      1                    1                  1
## 5 XANPAGTX0501_0…                      1                    1                  1
## 6 XANPAGTX0501_0…                      1                    1                  1
## 7 XANPAGTX0501_0…                      0                    0                  1
## 8 XANPAGTX0501_0…                      0                    0                  1
## 9 XANPAGTX0501_0…                      0                    0                  1
## # ℹ abbreviated name: ¹​`Apothecia / Vegetative (edge + center)`

• per functional group, how many of the lichen-upregulated genes are also apothecia-upregulated

lichen_upr<-read.delim2("../analysis_and_temp_files/08_dge_culture_lichen/upreg_in_lichen_sleuth.txt")
count_function<-ips_selected %>% filter(TranscriptID %in%lichen_upr$target_id) %>% group_by(Function) %>% summarize(total=n())
ips_selected %>% filter(TranscriptID %in% lichen_upr$target_id) %>%
  mutate(is_ap = ifelse(TranscriptID %in% apothecia_up,T,F)) %>%
  group_by(Function) %>% summarize(in_apothecia_up=sum(is_ap)) %>% left_join(count_function)

## Joining with `by = join_by(Function)`

## # A tibble: 19 × 3
##    Function                                  in_apothecia_up total
##    <chr>                                               <int> <int>
##  1 "Acetyl-CoA production and metabolism"                  1     4
##  2 "Carbohydrate storage"                                  1     1
##  3 "Cell division, proliferation and growth"               6    12
##  4 "Cell surface and cell wall proteins"                   2     9
##  5 "Cell wall biosynthesis "                               1     1
##  6 "Cell wall misc"                                        0     2
##  7 "Cell wall remodeling"                                  1     3
##  8 "Defense"                                               1     3
##  9 "G-proteins and GPCR"                                   0     1
## 10 "Lipid metabolism"                                      0     3
## 11 "Oxylipins"                                             0     2
## 12 "Protein ubiquitination"                                8    14
## 13 "RNA binding proteins"                                  4     5
## 14 "RNA interference"                                      3     3
## 15 "Ribosomal genes"                                       0     1
## 16 "Stress response genes"                                 0     1
## 17 "Transcription factors"                                 5    23
## 18 "Transporters"                                          0    27
## 19 "Velvet"                                                0     2

• None of lichen-upregulated transporters are apothecia-upregulated!
• Well, there is one protein we can id as a transporter that is upregulated in both, but in addition to that apothecia have 4 more that are not lichen upregulated!

compiled %>% filter(change=="apothecium", grepl("transporter",InterPro_new)) %>%
  filter(InterPro_new!="IPR004853 Sugar phosphate transporter domain",
         !grepl("ABC transporter-like",InterPro_new)) %>%
  select(target_id,InterPro_new,b,Comparison)  %>% 
  mutate(lichen_upregulated = ifelse(target_id %in%lichen_upr$target_id,T,F))

## # A tibble: 7 × 5
##   target_id              InterPro_new            b Comparison lichen_upregulated
##   <chr>                  <chr>               <dbl> <chr>      <lgl>             
## 1 XANPAGTX0501_001825-T1 IPR003663 Sugar/in… -1.06 Apothecia… FALSE             
## 2 XANPAGTX0501_010381-T1 IPR002293 Amino ac… -1.16 Apothecia… TRUE              
## 3 XANPAGTX0501_008594-T1 IPR036259 MFS tran… -1.04 Edge / Ap… FALSE             
## 4 XANPAGTX0501_000281-T1 IPR011701 Major fa… -1.28 Edge / Ap… FALSE             
## 5 XANPAGTX0501_001825-T1 IPR003663 Sugar/in… -1.40 Edge / Ap… FALSE             
## 6 XANPAGTX0501_009737-T1 IPR005829 Sugar tr… -1.54 Edge / Ap… FALSE             
## 7 XANPAGTX0501_010381-T1 IPR002293 Amino ac… -1.59 Edge / Ap… TRUE

Of the tisuue-DGE genes, what portion was lichen/culture DGE?

culture_upr<-read.delim2("../analysis_and_temp_files/08_dge_culture_lichen/upreg_in_culture_sleuth.txt")
compiled %>% mutate(lichen_cult = case_when(
  target_id %in% lichen_upr$target_id ~ "upreg_lichen" ,
  target_id %in% culture_upr$target_id ~ "upreg_culture",
  T ~ "non_dge"
)) %>% group_by(Comparison,change,lichen_cult) %>%
  summarize(n=n()) %>%
  pivot_wider(names_from=lichen_cult,values_from = n,values_fill = 0) %>%
  mutate(total= upreg_lichen+upreg_culture+non_dge)

## `summarise()` has grouped output by 'Comparison', 'change'. You can override
## using the `.groups` argument.

## # A tibble: 7 × 6
## # Groups:   Comparison, change [7]
##   Comparison                     change non_dge upreg_culture upreg_lichen total
##   <chr>                          <chr>    <int>         <int>        <int> <int>
## 1 Apothecia / Vegetative (edge … apoth…      56            17          177   250
## 2 Apothecia / Vegetative (edge … thall…       0             1            1     2
## 3 Center / Apothecia             apoth…      21             7           90   118
## 4 Center / Apothecia             centre       0             0            1     1
## 5 Edge / Apothecia               apoth…     121            25          225   371
## 6 Edge / Apothecia               edge         3             1            9    13
## 7 Edge / Center                  centre       1             1            7     9

• look at the culture-upregulated genes

compiled2 <- compiled %>% mutate(lichen_cult = case_when(
  target_id %in% lichen_upr$target_id ~ "upreg_lichen" ,
  target_id %in% culture_upr$target_id ~ "upreg_culture",
  T ~ "non_dge")) %>% 
  filter(lichen_cult=="upreg_culture")
compiled2 %>%  kable(format = "html", col.names = colnames(compiled2)) %>%
  kable_styling() %>%
  kableExtra::scroll_box(width = "100%", height = "400px")

Comparison	change	target_id	b	InterPro_new	CAZyme_new	Protease_new	KO	secreted_consensus	lichen_ortho	lichen_cult
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_007473-T1	-1.021407	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001223-T1	-1.064214	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_009599-T1	-1.070929	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001948-T1	-1.099351	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_006310-T1	-1.105045	IPR022137 Zinc finger protein, DUF3669 domain	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001643-T1	-1.215746	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001952-T1	-1.257146	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001950-T1	-1.259001	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001951-T1	-1.262779	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_001947-T1	-1.303157	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_002165-T1	-1.325802	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_002163-T1	-1.392989	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_002164-T1	-1.582652	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_009619-T1	-1.640066	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_003209-T1	-1.709229	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_009602-T1	-1.777131	NA	NA	NA		FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	apothecium	XANPAGTX0501_008109-T1	-2.159140	IPR004143 Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL), catalytic domain	NA	NA	K03800	FALSE	FALSE	upreg_culture
Apothecia / Vegetative (edge + center)	thallus	XANPAGTX0501_009376-T1	2.496154	IPR043141 Ribosomal protein L10-like domain superfamily	NA	NA	K02864	FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_002163-T1	-1.035184	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_001947-T1	-1.046488	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_001951-T1	-1.118810	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_003209-T1	-1.170208	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_009602-T1	-1.212068	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_009619-T1	-1.314627	NA	NA	NA		FALSE	FALSE	upreg_culture
Center / Apothecia	apothecium	XANPAGTX0501_008109-T1	-2.657633	IPR004143 Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL), catalytic domain	NA	NA	K03800	FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_005583-T1	-1.079779	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_000885-T1	-1.108859	IPR036305 RGS domain superfamily	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_010011-T1	-1.134493	IPR036013 Band 7/SPFH domain superfamily	NA	NA	K07192	FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009812-T1	-1.143459	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009785-T1	-1.191020	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009599-T1	-1.242828	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_003864-T1	-1.363940	IPR009075 Acyl-CoA dehydrogenase/oxidase C-terminal, IPR009100 Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain superfamily, IPR013786 Acyl-CoA dehydrogenase/oxidase, N-terminal, IPR036250 Acyl-CoA dehydrogenase-like, C-terminal, IPR037069 Acyl-CoA dehydrogenase/oxidase, N-terminal domain superfamily	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_007473-T1	-1.375934	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_007936-T1	-1.379176	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_005640-T1	-1.380432	IPR013087 Zinc finger C2H2-type	NA	NA		FALSE	TRUE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009761-T1	-1.387848	IPR001283 Cysteine-rich secretory protein-related, IPR014044 CAP domain, IPR018244 Allergen V5/Tpx-1-related, conserved site, IPR034120 None, IPR035940 CAP superfamily	NA	NA	K20412	TRUE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001951-T1	-1.498238	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009737-T1	-1.540774	IPR005829 Sugar transporter, conserved site, IPR011701 Major facilitator superfamily, IPR020846 Major facilitator superfamily domain, IPR036259 MFS transporter superfamily	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001948-T1	-1.662753	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001950-T1	-1.678963	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001949-T1	-1.732732	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001947-T1	-1.767840	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_002163-T1	-1.904022	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001643-T1	-1.910689	IPR001810 F-box domain, IPR032675 Leucine-rich repeat domain superfamily, IPR036047 F-box-like domain superfamily	NA	NA		FALSE	TRUE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_002165-T1	-1.959472	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_001952-T1	-2.002699	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009619-T1	-2.203533	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_003209-T1	-2.437600	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_009602-T1	-2.500879	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	apothecium	XANPAGTX0501_002164-T1	-2.527734	NA	NA	NA		FALSE	FALSE	upreg_culture
Edge / Apothecia	edge	XANPAGTX0501_006869-T1	1.099929	IPR008979 Galactose-binding-like domain superfamily, IPR017853 Glycoside hydrolase superfamily, IPR024655 Asl1-like, glycosyl hydrolase catalytic domain	GH128	NA		TRUE	FALSE	upreg_culture
Edge / Center	centre	XANPAGTX0501_001949-T1	-1.187615	NA	NA	NA		FALSE	FALSE	upreg_culture